U.S. patent number 5,763,269 [Application Number 08/384,476] was granted by the patent office on 1998-06-09 for recombinant infectious bovine rhinotrocheitis virus.
This patent grant is currently assigned to Syntro Corporation. Invention is credited to Mark D. Cochran, William P. MacConnell, Richard D. MacDonald, Meng-Fu Shih.
United States Patent |
5,763,269 |
Cochran , et al. |
June 9, 1998 |
Recombinant infectious bovine rhinotrocheitis virus
Abstract
The present invention provides a hybrid, nonprimate herpesvirus
comprising DNA which includes a sequence essential for viral
replication of the hybrid, nonprimate herpesvirus, at least a
portion of which is present in a sequence essential for replication
of a naturally-occurring nonprimate herpesvirus and at least one
foreign DNA sequence. Also provided is an attenuated, nonprimate
herpesvirus comprising DNA which includes a sequence essential for
viral replication of the attenuated, nonprimate herpesvirus, at
least a portion of which is present in a sequence essential for
replication of a naturally-occurring nonprimate herpesvirus, from
which at least a portion of a repeat sequence has been deleted.
Also provided are vaccines comprising the viruses of the invention
and methods of immunizing animals against various disease.
Inventors: |
Cochran; Mark D. (Carlsbad,
CA), Shih; Meng-Fu (San Diego, CA), MacConnell; William
P. (Cardiff, CA), MacDonald; Richard D. (San Diego,
CA) |
Assignee: |
Syntro Corporation (Lenexa,
KS)
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Family
ID: |
27574838 |
Appl.
No.: |
08/384,476 |
Filed: |
February 1, 1995 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
Issue Date |
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117633 |
Sep 7, 1993 |
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914057 |
Jul 13, 1992 |
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696262 |
Apr 30, 1991 |
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933107 |
Nov 20, 1986 |
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902877 |
Sep 2, 1986 |
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887140 |
Jul 17, 1986 |
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823102 |
Jan 27, 1986 |
5068192 |
Nov 26, 1991 |
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773430 |
Sep 6, 1985 |
4877737 |
Oct 31, 1989 |
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Current U.S.
Class: |
435/320.1;
435/235.1 |
Current CPC
Class: |
A61K
39/245 (20130101); C07K 14/005 (20130101); C12N
15/86 (20130101); A61K 39/12 (20130101); A61K
39/00 (20130101); A61K 2039/5254 (20130101); A61K
2039/5256 (20130101); C12N 2710/16722 (20130101); C12N
2710/16734 (20130101); C12N 2710/16743 (20130101); C12N
2720/12322 (20130101); C12N 2750/14322 (20130101); A61K
2039/545 (20130101); A61K 2039/57 (20130101) |
Current International
Class: |
C12N
15/40 (20060101); C12N 15/46 (20060101); C12N
15/86 (20060101); C12N 015/86 (); C12N
015/46 () |
Field of
Search: |
;435/172.1,172.3,320.1,69.1,91.1 |
References Cited
[Referenced By]
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EP |
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0141458 |
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EP |
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0162738 |
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EP |
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0176170 |
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EP |
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0216564 |
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EP |
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EP |
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EP |
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WO |
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WO |
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8704463 |
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Jul 1987 |
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WO |
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8901040 |
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Feb 1989 |
|
WO |
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|
Primary Examiner: Guzo; David
Attorney, Agent or Firm: White; John P.
Parent Case Text
The subject application is a continuation of U.S. Ser. No.
08/117,633, filed Sep. 7, 1993; now abandoned which is a
continuation of U.S. Ser. No. 07/914,057, filed Jul. 13, 1992, now
abandoned; which is a continuation of U.S. Ser. No. 07/696,262,
filed April 30, 1991, now abandoned; which is a continuation of
U.S. Ser. No. 06/933,107, filed Nov. 20, 1986, now abandoned; which
is a continuation-in-part of U.S. Ser. No. 06/902,877, filed Sep.
2, 1986, now abandoned which is a continuation-in-part of U.S. Ser.
No. 06/887,140, filed Jul. 17, 1986, now abandoned; which is a
continuation-in-part of U.S. Ser. No. 06/823,102, filed Jan. 27,
1986, now U.S. Pat. No. 5,068,192, issued Nov. 26, 1991, which is a
continuation-in-part of U.S. Ser. No. 06/773,430, filed Sep. 6,
1988, now U.S. Pat. No. 4,879,737, issued Oct. 31, 1989, which are
hereby incorporated by reference into the present application.
Claims
What is claimed is:
1. A recombinant infectious bovine rhinotracheitis virus comprising
a foreign DNA sequence encoding a polypeptide inserted into an
infectious bovine rhinotracheitis viral genome at a SacI site
within a 4.5Kb XhoI-HindIII subfragment of a Hind III K fraqment of
the infectious bovine rhinotracheitis viral genome, wherein
expression of the foreign DNA sequence is under the control of a
promoter located upstream of the foreign DNA sequence.
2. The recombinant infectious bovine rhinotracheitis virus of claim
1, wherein the polypeptide is an antigenic polypeptide.
3. The recombinant infectious bovine rhinotracheitis virus of claim
1 designated S-IBR-004.
4. The recombinant infectious bovine rhinotracheitis virus of claim
1, wherein the foreign DNA sequence is adapted for expression by an
inserted heterologous upstream herpesvirus promoter.
5. The recombinant infectious bovine rhinotracheitis virus of claim
2, wherein the antigenic polypeptide is bovine rotavirus
glycoprotein 38.
6. The recombinant infectious bovine rhinotracheitis virus of claim
4, wherein the inserted heterologous herpesvirus promoter is
selected from a group consisting of: the herpes simplex type 1 ICP4
protein promoter, the herpes simplex type I thymidine kinase
promoter, the pseudorabies immediate early gene promoter, the
pseudorabies glycoprotein X promoter, and the pseudorabies
glycoprotein 92 promoter.
7. A recombinant infectious bovine rhinotracheitis virus comprising
a foreign DNA sequence encoding a polypeptide inserted into an
infectious bovine rhinotracheitis viral genome at a non-essential
region within the HindIII K fragment of the infectious bovine
rhinotracheitis viral genome, wherein expression of the foreign DNA
sequence is under the control of a promoter located upstream of the
foreign DNA sequence.
8. The recombinant infectious bovine rhinotracheitis virus of claim
21, wherein the non-essential region is within a 4.5 Kb XhoI to
HindIII subfragment of the HindIII K fragment.
9. The recombinant infectious bovine rhinotracheitis virus of claim
1, wherein the non-essential region is between the SacI site and a
HindIII site within the 4.5 Kb XhoI to HindIII subfragment.
10. The recombinant infectious bovine rhinotracheitis virus of
claim 9, wherein the non-essential region is between the SacI site
and a XhoI site within the 4.5 Kb XhoI to HindIII subfragment.
Description
BACKGROUND OF THE INVENTION
Within this application several publications are referenced by
Arabic numerals within parentheses. Full citations for these
references may be found at the end of the specification immediately
preceding the claims. The disclosures of these publications in
their entirety are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
The advent of recombinant DNA techniques has made it possible to
manipulate the naturally occurring DNA sequences within an organism
(the genome) in order to change in some manner the functions of the
organism through genetic engineering. The present invention
concerns organisms defined as viruses that infect animals and
contain DNA as their genetic material; specifically viruses
belonging to the herpesvirus group (herpesviruses) (28). This group
of viruses comprise a number of pathogenic agents that infect and
cause disease in a number of target species: swine, cattle,
chickens, horses, dogs, cats, etc. Each herpesvirus is specific for
its host species, but they are all related in the structure of
their genomes, their mode of replication, and to some extent in the
pathology they cause in the host animal and in the mechanism of the
host immune response to the virus infection.
The types of genetic engineering that have been performed on these
herpesviruses consist of cloning parts of the virus DNA into
plasmids in bacteria, reconstructing the virus DNA while in the
cloned state so that the DNA contains deletions of certain
sequences, and furthermore adding foreign DNA sequences either in
place of the deletions or at sites removed from the deletions. The
usual method is to make insertions of the foreign DNA into the
viral sequences, although the foreign DNA could be attached to the
end of the viral DNA as well. One utility of the addition of
foreign sequences is achieved when the foreign sequence encodes a
foreign protein that is expressed during viral infection of the
animal. A virus with these characteristics is referred to as a
vector, because it becomes a living vector which will carry and
express the foreign protein in the animal. In effect it becomes an
elaborate delivery system for the foreign protein.
The prior art for this invention stems first from the ability to
clone and analyze DNA while in the bacterial plasmids. The
techniques that are available for the most part are detailed in
Maniatis et al. (1). This publication gives state-of-the-art
general recombinant DNA techniques.
The application of recombinant DNA techniques to animal viruses has
a relatively recent history from about 1980. The first viruses to
be engineered have been the smallest ones--the papovaviruses. The
viruses contain 3000-4000 base pairs (bp) of DNA in their genome.
Their small size makes analysis of their genomes relatively easy
and in fact most of the ones studied (SV40, polyoma, bovine
papilloma) have been entirely sequenced. Because these virus
particles are small and cannot accommodate much extra DNA, and
because their DNA is tightly packed with essential sequences (that
is, sequences required for replication), it has not been possible
to engineer these viruses as live vectors for foreign gene
expression. Their entire use in genetic engineering has been as
defective replicons for the expression of foreign genes in animal
cells in culture (roughly analogous to plasmids in bacterial
systems) or to their use in mixed populations of virions in which
wild type virus acts as a helper for the virus that has replaced an
essential piece of DNA with a foreign gene. The studies on
papovaviruses do not suggest or teach the concept of living virus
vectors as delivery systems for host animals.
The next largest DNA animal viruses are the adenoviruses. In these
viruses there is a small amount of nonessential DNA that can be
replaced by foreign sequences. The only foreign genes that seem to
have been expressed in adenoviruses are the T-antigen genes from
papovaviruses (2,3,4,5), and the herpes simplex virus thymidine
kinase gene (29). It is possible, given this initial success, to
envision the insertion of other small foreign genes into
adenoviruses. However the techniques used in adenoviruses do not
teach how to obtain the same result with herpesviruses. In
particular, these results do not identify the nonessential regions
in herpesviruses wherein foreign DNA can be inserted, nor do they
teach how to achieve the expression of the foreign genes in
herpesviruses, e.g. which promoter signals and termination signals
to use. Another group of animal viruses that have been engineered
are the poxviruses. One member of this group, vaccinia, has been
the subject of much research on foreign gene expression. Poxviruses
are large DNA-containing viruses that replicate in the cytoplasm of
infected cells. They have a structure that is very unique among
viruses--they do not contain any capsid that is based upon
icosahedral symmetry or helical symmetry. In theorizing on the
origin of viruses, the poxviruses are the most likely ones to have
originated from bacterial-like microorganisms through the loss of
function and degeneration. In part due to this uniqueness, the
advances made in the genetic engineering of poxviruses cannot be
directly extrapolated to other viral systems, including
herpesviruses. Vaccinia recombinant virus constructs have been made
in a number of laboratories that express the following inserted
foreign genes: herpes simplex virus thymidine kinase gene (6,7),
hepatitis B surface antigen (8,9,30), herpes simplex virus
glycoprotein D gene (8,30), influenza hemagglutinin gene (10, 11),
malaria antigen gene (12), and vesicular stomatitis glycoprotein G
gene (13). The general overall features of the vaccinia recombinant
DNA work are similar to the techniques used for all the viruses,
especially as they relate to the techniques in reference (1).
However in detail, the vaccinia techniques do not teach how to
engineer herpesviruses. Vaccinia DNA is not infectious, so the
incorporation of foreign DNA must involve an infection/transfection
step that is not appropriate to other viruses, and vaccinia has
unique stability characteristics that make screening easier. In
addition, the signal sequence used by promoters in vaccinia are
unique and will not work in other viruses. The utility of vaccinia
as a vaccine vector is in question because of its close
relationship to human smallpox and its known pathogenicity to
humans. The use of host-specific herpesviruses promises to be a
better solution to animal vaccination.
Among the herpesviruses, only herpes simplex of humans and, to a
limited extent, herpes saimiri of monkeys have been engineered to
contain foreign DNA sequences previous to this disclosure. The
earliest work on the genetic manipulation of herpes simplex virus
involved the rescue of temperature sensitive mutants of the virus
using purified restriction fragments of DNA (14). This work did not
involve cloning of the DNA fragments into the viral genome. The
first use of recombinant DNA to manipulate herpes simplex virus
involved cloning a piece of DNA from the L-S junction region into
the unique long region of the DNA, specifically into the thymidine
kinase gene (15). This insert was not a foreign piece of DNA,
rather it was a naturally occurring piece of herpesvirus DNA that
was duplicated at another place in the genome. This piece of DNA
was not engineered to specifically express any protein, and thus it
did not teach how to express protein in herpesviruses. The
manipulation of herpes simplex next involved the creation of
deletions in the virus genome by a combination of recombinant DNA
and thymidine kinase selection. The first step was to make a
specific deletion of the thymidine kinase gene (16). The next step
involved the insertion of the thymidine kinase gene into the genome
at a specific site, and then the thymidine kinase gene and the
flanking DNA at the new site were deleted by a selection against
thymidine kinase (17). In this manner herpes simplex alpha-22 gene
has been deleted (17). In the most recent refinement of this
technique, a 15,000 bp sequence of DNA has been deleted from the
internal repeat of herpes simplex virus (18).
The insertion of genes that encode protein into primate
herpesviruses have involved seven cases: the insertion of herpes
simplex glycoprotein C back into a naturally occurring deletion
mutant of this gene in herpes simplex virus (19); the insertion of
glycoprotein D of herpes simplex type 2 into herpes simplex type 1
(20), again with no manipulation of promoters since the gene is not
really `foreign`; the insertion of hepatitis B surface antigen into
herpes simplex virus under the control of the herpes simplex ICP4
promoter (21); and the insertion of bovine growth hormone into
herpes saimiri virus with an SV40 promoter that in fact didn't work
in that system (an endogenous upstream promoter served to
transcribe the gene) (22). Two additional cases of foreign genes
(chicken ovalbumin gene and Epstein-Barr virus nuclear antigen)
have been inserted into herpes simplex virus (31), and glycoprotein
X of pseudorabies virus has been inserted into herpes simplex virus
(33).
These limited cases of deletion and insertion of genes into
herpesviruses demonstrate that it is possible to genetically
engineer herpesvirus genomes by recombinant DNA techniques. The
methods that have been used to insert genes involve homologous
recombination between the viral DNA cloned on plasmids and purified
viral DNA transfected into the same animal cell. In aggregate this
is referred to as the homologous recombination technique. This
technique with minor modifications has been adaptable to other
herpesviruses that we have engineered. However, the extent to which
one can generalize the location of the deletion and the sites for
insertion of foreign genes is not obvious from these previous
studies. Furthermore, it is also not obvious that non-primate
herpesviruses are amenable to the same techniques as the primate
herpesviruses, and that one could establish a targeted approach to
the deletion, insertion, and expression of foreign genes.
One subject of this invention is a vaccine for pseudorabies virus
(herpesvirus suis, suid herpesvirus 1, or Aujesky's disease virus)
disease of swine. Swine are the natural host of pseudorabies virus
in which infection in older animals is commonly inapparent but may
be characterized by fever, convulsions, and death particularly in
younger animals. Pseudorabies also infects cattle, sheep, dogs,
cats, ferrets, foxes, and rats (39) where the infection usually
results in death. Death is usually preceded by intense pruritus,
mania, encephalitis, paralysis, and coma. Traditional live vaccines
are available for use in swine, but they are lethal for the other
animals. An improved vaccine for pseudorabies would induce a more
reliable immune response in swine, would be specifically attenuated
to be incapable of reversion to virulence, and would not cause
disease in other hosts.
Pseudorabies virus, an alpha-herpesvirus of swine, has a genome of
class D (23); that is it contains two copies of a single repeat
region, one located between the unique long and unique short DNA
region and one at the terminus of the unique short region (see FIG.
1). Herpes simplex virus is an alpha-herpesvirus with a class E
genome (28); that is it contains two copies of each of two repeats.
Herpes saimiri is a gamma-herpesvirus with a class B genome (28)
and thus is less related structurally to pseudorabies than is
herpes simplex virus.
Pseudorabies virus has been studied using the tools of molecular
biology including the use of recombinant DNA techniques. BamHI,
KpnI, and BgIII restriction maps of the virus genome have been
published (24, 27). DNA transfection procedures have been utilized
to rescue temperature sensitive and deletion mutants of the virus
by the homologous recombination procedure (24). There are two
examples of deletions that have been made in the pseudorabies virus
genome--one is a thymidine kinase gene deletion (25), also
disclosed in Pat. No. 4,514,497 entitled "Modified Live
Pseudorabies Viruses". This patent teaches thymidine kinase
deletions only and does not suggest other attenuating deletions,
nor does it suggest insertion of foreign DNA sequences. The other
reference involves the deletion of a small DNA sequence around a
HindIII restriction site in the repeat region (26) upon which
European Patent Publication No. 0141458, published on May 15, 1985,
corresponding to European Patent Application No. 84201474.8, filed
on Oct. 12, 1984 is based. This patent application does not teach
or suggest attenuating deletions nor does it teach or suggest the
insertion of DNA sequences into pseudorabies virus.
Other relevant pseudorabies literature disclosed herein, concerns
the presence of naturally-occurring deletions in the genome of two
vaccine strains of pseudorabies viruses (27). These deletions are
responsible, at least in part, for the attenuated nature of these
vaccines. Such naturally-occurring deletions do not teach methods
for making these deletions starting with wild type pseudorabies
virus DNA, nor do they suggest other locations at which to make
attenuating deletions. There are no examples of naturally-occurring
insertions of foreign DNA in herpesviruses.
Infectious bovine rhinotracheitis (IBR) virus, an alpha-herpesvirus
with a class D genome, is an important pathogen of cattle. It has
been associated with respiratory, ocular, reproductive, central
nervous system, enteric, neonatal and dermal diseases (39). Cattle
are the normal hosts of IBR virus, however it also infects goats,
swine, water buffalo, wildebeest, mink and ferrets. Experimental
infections have been established in muledeer, goats, swine, ferrets
and rabbits (40).
Conventional modified live virus vaccines have been widely used to
control diseases caused by IBR. These vaccine viruses may revert to
virulence, however. More recently, killed virus IBR vaccines have
been used, but their efficacy appears to be marginal.
IBR has been analyzed at the molecular level as reviewed in (41). A
restriction map of the genome is available in this reference, which
will aid in the genetic engineering of IBR according to the methods
provided by the present invention. No evidence has been presented
that IBR has been engineered to contain a deletion or an insertion
of foreign DNA.
Marek's disease virus (MDV) causes fowl paralysis, a common
lymphoproliferative disease of chickens. The disease occurs most
commonly in young chickens between 2 and 5 months of age. The
prominant clinical signs are progressive paralysis of one or more
of the extremeties, incoordination due to paralysis of legs,
drooping of the limb due to wing involvement, and a lowered head
position due to involvement of the neck muscles. In acute cases,
severe depression may result. In the case of highly oncogenic
strains, there is characteristic bursal and thymic atrophy. In
addition, there are lymphoid tumors affecting the gonads, lungs,
liver, spleen, kidney and thymus (39).
All chicks are vaccinated against MDV at one day of age to protect
the chick against MDV for its lifetime. One vaccine method for MDV
involves using turkey herpesvirus (HVT). It would be advantageous
to incorporate other antigens into this vaccination at one day of
age, but efforts to combine vaccines have not proven satisfactory
to date due to competition and immunosuppression between pathogens.
The multivalent vaccines engineered in this invention are a novel
way to simultaneously vaccinate against a number of different
pathogens.
A restriction map of both MDV (45) and HVT (36) are available in
the literature. There is no evidence to suggest that anyone has
successfully created a deletion or insertion of foreign DNA into
MDV or HVT prior to this disclosure.
Other herpesviruses contemplated to be amenable to these procedures
are feline herpesvirus (FHV), equine herpesvirus (EHV), and canine
herpesvirus (CHV). These pathogens cause disease in each of their
respective hosts. Feline herpesvirus causes feline rhinotracheitis,
an acute upper respiratory tract infection characterized by fever,
pronounced sneezing, nasal and lacrimal secretions, and depression.
The virus may cause corneal ulceration and abortion. The nasal
passages and turbinates show focal necrosis, and the tonsils are
enlarged and hemorrhagic. Equine herpesvirus causes
rhinopneumonitis, abortion, exanthema of the genitals and
occasionally neurologic disease. The acute disease is characterized
by fever, anorexia and a profuse, serous nasal discharge. The
neurologic symptoms, when present, consist of ataxia, weakness and
paralysis. Canine herpesvirus causes severe illness in young
puppies, where mortality may reach 80%. The disease is
characterized by viremia, anorexia, respiratory illness, abdominal
pain, vomiting and incessant crying. Generally, there is no fever.
The principal lesions are disseminated necrosis and hemorrhages in
the kidneys, liver and lungs.
The molecular biology of the feline, equine and canine
herpesviruses are in their initial phases. Partial restriction maps
are available for equine herpesvirus, and in progress in at least
one lab for the feline herpesvirus. Beyond this type of genome
analysis, no evidence for the deletion or insertion of foreign
genes into these viruses is available.
The present invention involves the use of genetically engineered
herpesviruses to protect animals against disease. It is not obvious
which deletions in herpesviruses would serve to attenuate the virus
to the proper degree. Even testing vaccine candidates in animal
models, e.g. mice, does not serve as a valid predictor of the
safety and efficacy of the vaccine in the target animal species,
e.g. swine.
SUMMARY OF THE INVENTION
The present invention provides a hybrid, nonprimate herpesvirus
comprising DNA which includes a sequence essential for viral
replication of the hybrid, non-primate herpesvirus, at least a
portion of which is present in a sequence essential for replication
of a naturally-occurring nonprimate herpesvirus and at least one
foreign DNA sequence.
Also provided is an attenuated, nonprimate herpesvirus comprising
DNA which includes a sequence essential for viral replication of
the attenuated, nonprimate herpesvirus, at least a portion of which
is present in a sequence essential for replication of a
naturally-occurring nonprimate herpesvirus, from which at least a
portion of a repeat sequence has been deleted.
The present invention further provides an attenuated, hybrid,
nonprimate herpesvirus. This virus comprises DNA which includes a
sequence essential for viral replication of the attenuated, hybrid,
nonprimate herpesvirus, at least a portion of which is present in a
sequence essential for replication of a naturally-occurring
nonprimate herpesvirus and at least one foreign DNA sequence.
BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A AND 1B DETAILS OF WILD
TYPE SHOPE STRAIN PRV
1A. Diagram of PRV genomic DNA showing the unique long region (UL),
the unique short region (US), the internal repeat region (IR), and
the terminal repeat region (TR).
1B. BamHI restriction enzyme map of PRV. Fragments are numbered in
order of decreasing size.
FIGS. 2A-2C Details of S-PRV-004 Construction and Map Data
2A. Detailed map of BamHI #8' and #8. The location of the internal
repeat (IR) region is shown.
2B. Detailed map of BamHI #8'-TK-8 fragment ultimately present in
the recombinant virus.
2C. Diagram of the S-PRV-004 DNA genome showing the location of the
HSV-1 TK gene inserted into the junction region between the UL and
IR regions.
Restriction Enzyme Legend: B=BamHI; K=KpnI; N=NdeI; P=PvuII;
S=StuI.
FIGS. 3A-3C Details of S-PRV-005 Construction and Map Data.
3A. Detailed map of BamHI #5. The HSV-1 TK gene fused to the HSV-1
ICP4 promoter is shown on a PvuII fragment.
3B. Detailed map of BamHI #5 after the insertion of the TK gene
construct.
3C. Diagram of the S-PRV-005 DNA genome showing the location of the
TK gene inserted into both copies of BamHI #5 in the repeat region
of the genome and the creation of new deletions.
Restriction Enzyme Legend: B=BamHI; H=HindIII; Hp=HpaI; K=KpnI;
P=PvuII; X=XbaI.
FIGS. 4A-4D Construction of the Foreign DNA Insert Used in
S-PRV-010.
4A. Diagram of the relevant portion of pJF751 that contains the lac
Z (beta-galactosidase) gene. The position of the TAA termination
codon for the polypeptide is indicated.
4B. Diagram of the promoter sequence from the HSV-1 TK gene.
4C. Diagram of the RsaI fragment of the TK gene now with BamHI
modified ends.
4D. Diagram of the final plasmid that contained the lac Z gene
fused to the HSV-1 TK promoter.
Restriction Enzyme legend: B=BamHI; Ba=BalI; Bc=BclI; Bg=BglII;
H=HindIII; Ha=HaeIII; N=NdeI; R=RsaI; X=XbaI.
FIGS. 5A-5C Details of S-PRV-010 Construction and Map Data.
5A. Detailed map of BamHI #5. The lac Z gene (beta-galactosidase)
fused to the HSV-1 TK promoter is shown on an XbaI fragment (see
FIGS. 4A-4D). The position of the deletion in S-PRV-002 is
shown.
5B. Detailed map of BamHI #5 after the insertion of the lac Z gene
construct.
5C. Diagram of the S-PRV-010 genome DNA showing the location of the
lac Z gene into both copies of BamHI #5 in the repeat region of the
genome.
Restriction Enzyme Legend: B=BamHI; Bc=BclI; H=HindIII; Hp=HpaI;
K=KpnI; N=NdeI; X=XbaI.
FIGS. 6A-6C Details of S-PRV-007 Construction and Map Data.
6A. Detailed map of BamHI #5 from S-PRV-005.
6B. Detailed map of BamHI #5 after the substitution of the TK gene
with the swine rotavirus gp38 gene.
6C. Diagram of the S-PRV-007 DNA genome showing the location of the
gp38 gene inserted into both copies of BamHI #5 in the repeat
regions of the genome.
Restriction Enzyme Legend: B=BamHI; H=HindIII; K=KpnI. FIG. 7
Construction of the Foreign DNA Insert Used in S-PRV-007.
FIGS. 8A-8C Details of S-PRV-012 Construction and Map Data.
8A. Detailed map of PRV extending from BamHI #10 through BamHI
#7.
8B. Detailed map of PRV extending from BamHI #10 through BamHI #7
after the insertion of the TK gene into the recombinant virus.
8C. Diagram of the S-PRV-012 DNA genome showing the location of the
TK gene inserted into the gpX region and the creation of a deletion
that removes most of the coding region of the gpX gene and renders
the virus unable to synthesize the gpX polypeptide.
Restriction Enzyme Legend: B=BamHI; K=KpnI; N=NdeI; P=PvuII;
Ps=PstI; S=StuI.
FIGS. 9A-9E Details of S-PRV-013. S-PRV-014, and S-PRV-016
Construction and Map Data.
9A. Detailed map of PRV extending from BamHI #10 through BamHI
#7.
9B. Detailed map of PRV extending from BamHI #10 through BamHI #7
after the insertion of the lac Z gene into the recombinant
virus.
9C. Diagram of the S-PRV-013 DNA genome showing the location of the
lac Z gene inserted into the gpX region and the creation of a
deletion that removed most of the coding region of the gpX gene and
rendered the virus unable to synthesize the gpX polypeptide. Other
deletions in the TK region and repeat regions are shown by
(.tangle-solidup.).
9D. Diagram of the S-PRV-014 DNA genome showing the location of the
lac Z gene inserted into the gpX region and the creation of a
deletion that removed most of the coding region of the gpX gene and
rendered the virus unable to synthesize the gpX polypeptide. There
are no other deletions in this virus.
9E. Diagram of the S-PRV-016 DNA genome showing the location of the
lac Z gene inserted into the gpX region and the creation of a
deletion that removed most of the coding region of the gpX gene and
rendered the virus unable to synthesize the gpX polypeptide. Other
deletions in the repeat regions are shown by
(.tangle-solidup.).
Restriction Enzyme Legend: B=BamHI; Ba=BalI; K=KpnI; N=NdeI;
Ps=PstI; S=StuI.
FIG. 10A and 11B Swine rotavirus gp38 Gene Sequence in pSY565.
FIG. 11A and 11B Swing parvovirus B gene sequence in pSY875.
FIG. 12 Swine parvovirus B gene construction with signal
sequence
A. pSY864 which contains the B gene from AccI at nucleotide #391 to
RsaI site at nucleotide #2051 cloned between the BamHI site in
BamHI #10 and the NdeI site in BamHI #7.
B. pSY957 which contains the SailI fragment from pSY864 cloned into
a polylinker in pSP65 so that XbaI sites flank the insert.
Legend: pSP=E. coli plasmid; PRV=pseudorabies virus DNA;
PPV=porcine parvovirus DNA; Pv=PvuII; RV=EcoRV; Ps=PstI; B=BamHI;
A=AccI; R=RsaI; N=NdeI; Sa=SalI; Sm=SmaI; S=StuI; X=XbaI; gpX
pro=glycoprotein X promoter; gpX pA=glycoprotein X polyadenylation
signal sequences.
FIG. 13A-13 C Details of S-PRV-020 Construction and Map Data
13A. Detailed map of PRV extending from BamHI #10 through BamHI #7
showing the parvovirus B gene that will replace the gpX gene.
13B. Detailed map of PRV from BamHI #10 through BamHI #7 after the
insertion of the swine parvovirus B gene in place of the gpX
gene.
13C. Diagram of the S-PRV-020 genome showing the location of the
swine parvovirus B gene inserted into the gpX region of PRV.
Restriction Enzyme Legend: B=BamHI; Ps=PstI;
Sa=SalI; N=NdeI; S=StuI; A=AccI; R=RsaI.
FIG. 14A-14C Details of S-PRV-025 construction and map data
14A. Region of S-PRV-002 starting virus showing BamHI #5 fragment.
The parvovirus B gene XbaI fragment from pSY957 is diagrammed below
showing how it will be inserted into the XbaI site by direct
ligation.
14B. Region of BamHI #5 after insertion of the parvovirus B
gene.
14C. Location of the parvovirus B gene inserted into both copies of
the repeat in S-PRV-025.
Legend: B=BamHI; H=HindIII; X=XbaI; S=SalI; pA=glycoprotein X
polyadenylation signal sequences; UL=unique long region; US=unique
short region; IR=internal repeat region; TR=terminal repeat
region.
FIG. 15A-15C Details of SPRV-029 Construction and Map Data
15A. Detailed map of PRV extending from BamHI #10 through BamHI #7
showing the lac Z gene that will replace the gpX gene.
15B. Detailed map of PRV extending from BamHI #8' through BamHI #8
at the junction of the unique long region and the internal repeat
region (IR). The lac Z gene as a SalI fragment will replace the DNA
between the StuI sites bracketing the junction.
15C. Diagram of the S-PRV-029 genome showing the locations of the
lac Z genes in the gpX region and the junction region.
Restriction Enzyme Legend: B=BamHI; Ps=PstI;
Sa=SalI; N=NdeI; S=StuI; Ba=BalI; K=KpnI.
FIG. 16 Restriction Map of Deleted S-IBR-002 EcoRI B Fragment and
EcoRI F Fragment.
An 800 bp deletion including EcoRV and BglII restriction sites was
mapped in both repeat fragments.
FIG. 17 Construction of Recombinant S-IBR-004 Virus.
S-IBR-004 is an IBR recombinant virus carrying an inserted foreign
gene (NEO) under the control of the PRV gpX promoter. A new XbaI
site was created at the small unique region and the original SacI
site was deleted.
FIG. 18 Construction of Recombinant S-IBR-008 Virus.
S-IBR-008 is a recombinant IBR virus that has a bovine rota
glycoprotein gene and the plasmid vector inserted in the XbaI site
on the unique long region. A site specific deletion was created at
the [SacI] site due to the loss of NEO gene in the small unique
region.
FIG. 19A-19C Details of HVT Construction and Map Data
19A. BamHI restriction fragment map of HVT. Fragments are numbered
in order of decreasing size; letters refer to small fragments whose
comparative size has not been determined.
19B. BamHI #16 fragment showing location of beta-galactosidase gene
insertion in S-HVT-001.
19C. BamHI #19 fragment showing location of beta-galactosidase gene
insertion.
Legend: B=BamHI; X=XhoI; H=HindIII; P=PstI; S =SalI; N=NdeI;
R=EcoRI.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a hybrid, nonprimate herpesvirus
comprising DNA which includes a sequence essential for viral
replication of the hybrid, nonprimate herpesvirus, at least a
portion of which is present in a sequence essential for replication
of a naturally-occurring nonprimate herpesvirus and at least one
foreign DNA sequence. The sequence essential for viral replication
of the hybrid, nonprimate herpesvirus may be derived from a
naturally-occurring nonprimate herpesvirus.
The foreign DNA sequence may be adapted for expression in a host
and encode an amino acid sequence. In one embodiment of the
invention, the foreign DNA sequence is adapted for expression by a
herpesvirus promoter. The herpesvirus promoter may be an endogenous
upstream herpesvirus promoter or an inserted upstream herpesvirus
promoter. Examples of such herpesvirus promoters include, but are
not limited to, the herpes simplex type ICP4 protein promoter, the
herpes simplex type I thymidine kinase promoter, the pseudorabies
thymidine kinase promoter, the pseudorabies immediate early gene
promoter, the pseudorabies glycoprotein X promoter or the
pseudorabies glycoprotein 92 promoter.
The amino acid sequence encoded by the foreign DNA sequence may be
a polypeptide. Furthermore, the polypeptide may be a protein. In
one embodiment of the invention, the protein, when expressed in the
host, is antigenic. In a further embodiment of the invention, the
protein is swine rotavirus glycoprotein 38. In yet another
embodiment of the invention, the protein is bovine rotavirus
glycorprotein 38. In yet a further embodiment of the invention, the
protein is swine parvovirus B capsid protein.
The hybrid, nonprimate herpesvirus may comprise DNA of which at
least a portion is present in a sequence essential for replication
of a naturally-occurring alpha-herpesvirus. The alpha-herpesvirus
may be a pseudorabies virus, infectious bovine rhinotracheitis
virus, equine herpesvirus I, feline herpesvirus I or canine
herpesvirus I. Additionally the alpha-herpesvirus may be a class D
herpesvirus. The class D herpesvirus may be pseudorabies virus,
infectious bovine rhinotracheitis virus, equine herpesvirus I,
feline herpesvirus I or canine herpesvirus I. In one embodiment of
the invention, the alpha-herpesvirus is an infectious bovine
rhinotracheitis virus and the foreign DNA encodes the Escherichia
coli neomycin resistance gene. This foreign DNA sequence may also
be under the control of an inserted pseudorabies virus glycoprotein
X promoter. Such a virus has been constructed, designated
S-IBR-004, and deposited on May 23, 1986 with the American Type
Culture Collection (ATCC), 12301 Parklawn Drive, Rockville,
Maryland 20852 under Accession No. VR 2134.
In another embodiment of the invention, the alpha-herpesvirus is an
infectious bovine rhinotracheitis virus with a deletion in the
unique short sequence. Furthermore, the foreign DNA sequence may
encode the bovine rotavirus glycoprotein 38 gene. This virus,
designated S-IBR-008, has been constructed and deposited Jun. 16,
1986 with the ATCC under Accession No. VR 2141.
Additionally the hybrid, nonprimate herpesvirus may comprise DNA of
which at least a portion is present in a sequence essential for
replication of a naturally-occurring gamma-herpesvirus. The
gamma-herpesvirus may be Marek's disease virus or herpesvirus of
turkeys. Moreover the gamma-herpesvirus may be a class E
herpesvirus. The class E herpesvirus may be Marek's disease virus
or herpesvirus of turkeys.
Also provided is an attenuated, nonprimate herpesvirus comprising
DNA which includes a sequence essential for viral replication of
the attenuated, nonprimate herpesvirus, at least a portion of which
is present in a sequence essential for replication of a
naturally-occurring nonprimate herpesvirus, from which at least a
portion of a repeat sequence has been deleted. The sequence
essential for viral replication of the attenuated, nonprimate
herpesvirus may be derived from a naturally-occurring nonprimate
herpesvirus.
The deleted portion of the repeat sequence may include a portion of
a repeat sequence other than a junction region or may include a
junction region. Additionally, the deleted portion of the repeat
sequence may comprise a nonessential sequence of one repeat
sequence or both repeat sequences. Furthermore at least a portion
of the essential sequence of a repeat may be deleted. In one
embodiment of the invention, one entire repeat may be deleted.
Moreover, a sequence not located within a repeat may additionally
be deleted. In one embodiment of the invention the deleted sequence
not located within a repeat is at least a portion of a gene.
The attenuated nonprimate herpesvirus may comprise DNA at least a
portion of which is present in a sequence essential for replication
of a naturally-occurring alpha-herpesvirus. The alpha-herpesvirus
may be a pseudorabies virus, infectious bovine rhinotracheitis
virus, equine herpesvirus I, feline herpesvirus I or canine
herpesvirus I. Additionally, the alpha-herpesvirus may be a class D
herpesvirus. The class D herpesvirus may be a pseudorabies virus,
infectious bovine rhinotracheitis virus, equine herpesvirus I,
feline herpesvirus I or canine herpesvirus I. In one embodiment of
the invention, the alpha-herpesvirus is an infectious bovine
rhinotracheitis virus. In another embodiment of the invention, the
attenuated, nonprimate herpesvirus comprises an infectious bovine
rhinotracheitis virus from which has been deleted at least a
portion of both repeat sequences. This virus has been constructed,
designated S-IBR-002, and deposited under ATCC Accession No. VR
2140.
Further provided is an attenuated, hybrid, nonprimate herpesvirus
comprising DNA which includes a sequence essential for viral
replication of the attenuated, hybrid, nonprimate herpesvirus, at
least a portion of which is present in a sequence essential for
replication of a naturally-occurring nonprimate, herpesvirus and at
least one foreign DNA sequence. The sequence essential for viral
replication of the attenuated, hybrid, nonprimate virus may be
derived from a naturally-occurring nonprimate herpesvirus.
Furthermore, at least a portion of a repeat sequence of the
attenuated, hybrid, nonprimate herpesvirus may be deleted.
The foreign DNA sequence may be adapted for expression in a host
and encode an amino acid sequence. Additionally, the foreign DNA
sequence may be adapted for expression by a herpesvirus promoter.
The herpesvirus promoter may be an endogenous upstream promoter or
an inserted upstream herpesvirus promoter. The herpesvirus promoter
may be the herpes simplex type ICP4 protein promoter, the herpes
simplex type I thymidine kinase promoter, the pseudorabies
immediate early gene promoter, the pseudorabies glycoprotein X
promoter or the pseudorabies glycoprotein 92 promoter.
The amino acid sequence encoded by the foreign DNA sequence may be
a polypeptide. Additionally the polypeptide may be a protein.
Furthermore the protein, when expressed in a host, may be
antigenic. In one embodiment of the invention the protein is swine
rotavirus glycoprotein 38. In another embodiment, the protein is
bovine rotavirus glycoprotein 38. In a further embodiment of the
invention, the protein is swine parvovirus B capsid protein.
The attenuated, hybrid, nonprimate herpesvirus may comprise DNA, at
least a portion of which is present in a sequence essential for
replication of a naturally-occurring alpha-herpesvirus. The
alpha-herpesvirus may be a pseudorabies virus, infectious bovine
rhinotracheitis virus, equine herpesvirus I, feline herpesvirus I
or canine herpesvirus I. Additionally, the alpha-herpesvirus may be
a class D herpesvirus. The class D herpesvirus may be pseudorabies
virus, infectious bovine rhinotracheitis virus, equine herpesvirus
I, feline herpesvirus I or canine herpesvirus I.
Furthermore the attenuated, hybrid, nonprimate herpesvirus may
comprise DNA, at least a portion of which is present in a sequence
essential for replication of a naturally-occurring
gamma-herpesvirus. The gamma-herpesvirus may be Marek's disease
virus or herpesvirus of turkeys. Additionally the gamma-herpesvirus
may be a class E herpesvirus. The class E herpesvirus may be
Marek's disease virus or herpesvirus of turkeys.
The present invention also provides a vaccine useful for immunizing
an animal against a herpesvirus disease. This vaccine comprises an
effective immunizing amount of a hybrid, nonprimate herpesvirus of
the present invention and a suitable carrier.
Also provided is a multivalent vaccine useful for immunizing an
animal against at least one pathogen. This vaccine comprises an
effective immunizing amount of a hybrid, nonprimate herpesvirus of
the present invention which includes a foreign DNA sequence
encoding a protein which, when expressed in the host, is antigenic
and a suitable carrier.
Furthermore, the present invention provides a vaccine useful for
immunizing an animal against a herpesvirus disease which comprises
an effective immunizing amount of an attenuated, nonprimate
herpesvirus provided by the invention and a suitable carrier.
Another vaccine useful for immunizing an animal against a
herpesvirus disease is also provided. This vaccine comprises an
effective immunizing amount of an attenuated, hybrid, nonprimate
herpesvirus of the present invention and a suitable carrier.
Moreover, a multivalent vaccine useful for immunizing an animal
against at least one pathogen is provided. This vaccine comprises
an effective immunizing amount of an attenuated, hybrid, nonprimate
herpesvirus which includes at least one foreign DNA sequence
encoding a protein which, when expressed in the host, is antigenic
and a suitable carrier.
Methods of immunizing animals against herpesvirus diseases and
methods of immunizing an animal against at least one pathogen are
provided. These methods comprise administering to the animal a
suitable dose of a vaccine of the present invention. The animals
which may be immunized include, but are not limited to, bovine
animals, sheep and goats.
Methods of identifying the hybrid, nonprimate herpesviruses are
provided. In one embodiment of the invention, the foreign DNA
sequence in the virus is detected. In another embodiment of the
invention, the presence of the expressed polypeptide in the host
animal or host cell is detected. In yet another embodiment of the
invention, the presence of the expressed protein in the host animal
or host cell is detected. Furthermore, methods of identifying an
attenuated, hybrid, nonprimate herpesvirus of the invention are
provided. In one embodiment of the invention, the foreign DNA
sequence is detected. In another embodiment of the invention, the
presence of the expressed polypeptide in the host animal or host
cell is detected. In yet a third embodiment of the invention, the
presence of the expressed protein in the host animal or host cell
is detected.
The present invention further provides a method of producing in an
animal a gene product for purposes other than immunization. This
method comprises administering to the animal a suitable quantity of
a hybrid, nonprimate herpesvirus of the present invention which
includes a foreign DNA sequence adapted for expression in a host,
the foreign DNA sequence of which expresses the gene product.
Additionally, a gene product may be produced in an animal for
purposes other than immunization by administering to the animal a
suitable quantity of an attenuated, hybrid, nonprimate herpesvirus
which includes a foreign DNA sequence adapted for expression in a
host, the foreign DNA sequence of which expresses the gene
product.
Methods of preparing an attenuated, hybrid, nonprimate herpesvirus
of the present invention are also provided. One method comprises
isolating naturally-occurring nonprimate herpesvirus viral DNA and
using restriction enzyme digestion to produce DNA restriction
fragments. These restriction fragments are purified by agarose gel
electrophoresis to obtain specific DNA fragments which are treated
with appropriate enzymes, known to those skilled in the art, to
produce modified viral DNA fragments. These modified DNA fragments
are capable of binding to bacterial plasmid DNA sequences. Suitable
bacterial plasmids are separately treated with appropriate
restriction enzymes, known to those skilled in the art, to produce
bacterial plasmid DNA sequences capable of binding to modified
viral DNA fragments. These bacterial plasmid sequences are then
combined with the modified viral DNA fragments under suitable
conditions to allow the viral DNA to bind the bacterial DNA and
form a viral-bacterial plasmid.
The viral-bacterial DNA plasmid is then mapped by restriction
enzymes to generate a restriction map of the viral DNA insert. The
viral-bacterial DNA plasmid is then treated with a restriction
enzyme known in the art to cause at least one deletion in the viral
DNA sequence of the viral-bacterial DNA plasmid. This plasmid,
containing at least one deletion in the viral DNA sequence, is
transfected with naturally-occurring nonprimate herpes viral DNA
into animal cells. The animal cells are maintained under suitable
conditions to allow the naturally-occurring nonprimate herpesviral
DNA to regenerate herpesviruses and a small percent of viruses
which have recombined with the viral-foreign DNA sequence of the
viral-bacterial-foreign DNA plasmid. Some of these recombined
viruses have deletions in their genome as a result of deletions in
the viral DNA insert of the plasmid. The viruses are identified and
subsequently plaque purified away from the undesired viruses.
In another embodiment of the invention, naturally-occurring
nonprimate herpes viral DNA is isolated and digested with
appropriate restriction enzymes to produce viral restriction
fragments. Separately, foreign DNA is digested with appropriate
enzymes to produce foreign DNA restriction fragments. The foreign
DNA restriction fragments are mixed with the viral DNA restriction
fragments under suitable conditions so as to allow the fragments to
join together to produce viral-foreign DNA fragments. Animal cells
are transfected with the viral-foreign DNA fragments and maintained
under suitable conditions so as to allow the foreign DNA fragments
to regenerate herpesviruses and a small percent of viruses which
have included foreign DNA fragments into their genome.
Herpesviruses which have included desired foreign DNA fragments
into their genome are identified and plaque purified away from
undesired herpesviruses.
MATERIALS AND METHODS
GROWTH OF HERPESVIRUS IN TISSUE CULTURE. All of the herpesviruses
under discussion were grown in tissue culture cells. Unless
otherwise noted, the cells used were: Vero cells for PRV; MDBK
cells for IBR; CEF cells for HVT; Crandall feline kidney cells for
FHV. Vero cells are suitable for EHV and MDCK cells are suitable
for CHV.
PREPARATION OF HERPESVIRUS STOCK SAMPLES. Herpesvirus stock samples
were prepared by infecting tissue culture cells at a multiplicity
of infection of 0.01 PFU/cell in Dulbecco's Modified Eagle Medium
(DMEM) containing 2 mM glutamine, 100 units/ml penicillin, 100
units/ml streptomycin (these components were obtained from Irvine
Scientific or equivalent supplier, and hereafter are referred to as
complete DME medium) plus 1% fetal bovine serum. After cytopathic
effect was complete, the medium and cells were harvested and the
cells were pelleted at 3000 rpm for 5 minutes in a clinical
centrifuge. For all herpesviruses except HVT, the cells were
resuspended in 1/10 the original volume of medium, and an equal
volume of 2 times autoclaved skim milk (9% skim milk powder in
H.sub.2 O wgt/vol) was added. The virus sample was frozen and
thawed 2 times, aliquoted, and stored frozen at -70.degree. C. The
titer was usually about 108 plaque forming units per ml. For HVT,
infected cells were resuspended in complete medium containing 20%
fetal bovine serum, 10% DMSO and stored frozen at -70.degree.
C.
PREPARATION OF HERPESVIRUS DNA. For herpesvirus DNA preparation, a
confluent monolayer of tissue culture cells in a 25 cm.sup.2 flask
or a 60 mm petri dish was infected with 100 microliters of virus
sample in 1 ml medium. Adsorption proceeded for 1-2 hours at 37
.degree. C. in a humidified incubator with 5% CO.sub.2 in air.
After adsorption, 4 mls of complete DME medium plus 1% fetal bovine
serum were added. After overnight incubation, or when the cells
were showing 100% cytopathic effect, the cells were scraped into
the medium with a cell scraper (Costar brand). The cells and medium
were centrifuged at 3000 rpm for 5 minutes in a clinical
centrifuge. The medium was decanted, and the cell pellet was gently
resuspended in 0.5 ml solution containing 0.01M Tris pH 7.5, 1 mM
EDTA, and 0.5% Nonidet P-40 (NP40, an ionic detergent comprising an
octyl phenol ethylene oxide condensate containing an average of 9
moles ethylene oxide per molecule, purchased from Sigma Chemical
Co., St. Louis, MO.). The sample was incubated at room temperature
for 10 minutes. Ten microliters of a stock solution of RNase A
(Sigma) were added (stock was 10 mg/ml, boiled for 10 minutes to
inactivate DNAase). The sample was centrifuged for 5 minutes at
3000 rpm in a clinical centrifuge to pellet nuclei. The DNA pellet
was removed with a pasteur pipette or wooden stick and discarded.
The supernatant fluid was decanted into a 1.5 ml Eppendorf tube
containing 25 microliters of 20% sodium dodecyl sulfate (Sigma) and
25 microliters proteinase-K (10 mg/ml; Boehringer Mannheim
supplier). The sample was mixed and incubated at 37.degree. C. for
30-60 minutes. An equal volume of water-saturated phenol was added
and the sample was mixed on a vortex mixer for 5 minutes. The
sample was centrifuged in an Eppendorf minifuge for 5 minutes at
full speed. The upper aqueous phase was removed to a new Eppendorf
tube, and two volumes of -20.degree. C. absolute ethanol were added
and the tube put at -20 .degree. C. for 30 minutes to precipitate
nucleic acid. The sample was centrifuged in an Eppendorf centrifuge
at 4.degree. C. for 5 minutes. The supernatant was decanted, and
the pellet was washed one time with cold 80% ethanol. The pellet
was dried in a lyophilizer, and rehydrated in 17 microliters
H.sub.2 O. For the preparation of larger amounts of DNA, the
procedure was scaled up to start with a 850 cm.sup.2 roller bottle
of Vero cells. The DNA was stored in H.sub.2 O or in 0.01 M Tris pH
7.5, 1 mM EDTA at -20 .degree. C.
PHENOL EXTRACTION. Phenol extraction was performed on any
convenient volume of DNA sample, typically between 100 microliters
to 1 ml. The DNA sample was diluted in 0.lM Tris pH 7.5, 1 mM EDTA
and an equal volume of water saturated phenol was added. The sample
was mixed briefly on a vortex mixer and placed on ice for 3
minutes. After centrifugation for 3 minutes in a microfuge, the
aqueous layer was removed to a new tube and was precipitated by
ethanol.
ETHANOL PRECIPITATION. DNA in a sample was concentrated by ethanol
precipitation. To the DNA sample were added 1/10 volume of 3M
sodium acetate, pH 7.5 and 3 volumes of cold ethanol. The DNA was
precipitated for 30 minutes at -70 .degree. C. or overnight at -20
.degree. C. and then pelleted by centrifugation in the microfuge
for 15 minutes at 4.degree. C. The pellet was washed once with 200
microliters of cold 80% ethanol and pelleted again for 10 minutes
at 4.degree. C. After air drying or lyophilization, the pellets
were resuspended in the appropriate buffer or H.sub.2 O.
RESTRICTION ENZYME DIGESTION. DNA was cut by restriction enzymes
using the buffer recommended by the manufacturer (International
Biotechnologies Inc., New Haven, CT. (IBI), Bethesda Research
Laboratories, Bethesda, MD. (BRL), and New England Biolabs,
Beverly, MA). Whenever possible, the concentration of DNA was kept
below 1 microgram/50 microliters. Incubation was at 37 .degree. C.
for 1-4 hours.
AGAROSE GEL ELECTROPHORESIS OF DNA. To visualize the restriction
pattern of the DNA, 5 microliters of loading buffer (5X
electrophoresis buffer, 0.01% bromphenol blue dye, 50 mM EDTA, and
50% glycerol) were added. The sample was loaded into a lane in a
horizontal submarine electrophoresis unit containing a 0.6% agarose
gel. The electrophoresis buffer was 40 mM Tris, 10 mM EDTA,
adjusted to pH 7.8 with acetic acid, and with or without 0.5
micrograms/ml ethidium bromide. The gel was run at 40-50V for 18
hours, and the gel was removed and stained with 0.5 micrograms/ml
ethidium bromide for 30 minutes. The DNA bands were visualized on a
long wavelength UV transilluminator.
PHOSPHATASE TREATMENT OF DNA. Phosphatase treatment of DNA was
performed by adding 1 microliter (25 units) of calf intestinal
phosphatase (Boehringer Mannheim) directly to the restriction
enzyme digestion reactions and continuing the incubation for 30
minutes at 37.degree. C. The phosphatase was inactivated for 60
minutes at 65.degree. C prior to phenol extraction.
POLYMERASE FILL-IN REACTION. DNA was resuspended in buffer
containing 50 mM Tris pH 7.4, 50 mM KC1, 5 mM MgCl.sub.2, and 400
micromolar each of the four deoxynucleotides. Ten units of Klenow
DNA polymerase (BRL) were added and the reaction was allowed to
proceed for 15 minutes at room temperature. The DNA was then phenol
extracted and ethanol precipitated as above.
EXONUCLEASE RESECTION REACTION. DNA was resuspended in 100
microliters of 60 mM Tris pH 8.0, 0.66 mM MgCl.sub.2, 1 mM
beta-mercaptoethanol. The sample was warmed to 30.degree. C. for 5
minutes, and 10 units of lambda exonuclease III (BRL) were added.
At frequent time intervals (e.g. every 2.5 minutes), 10 microliter
aliquots were diluted into 100 microliters of 30 mM sodium acetate
pH 4.5, 250 mM NaCl, 1 mM ZnSO.sub.4, 4 micrograms/100 microliters
yeast tRNA, 30 units/ 100 microliters S1 nuclease. After 45 minutes
at 30.degree. C., 15 microliters of stop buffer consisting of 625
mM Tris pH 9.0, 150 mM EDTA, 1% SDS were added. The samples were
then phenol extracted and ethanol precipitated as above. The DNA
digestion products were then analyzed and purified by agarose gel
electrophoresis.
PHENOL EXTRACTION OF DNA FROM AGAROSE. DNA bands cut from low
melting point agarose gels were diluted to less than 0.5% agarose
to a final concentration of 0.3 M sodium acetate. The samples were
heated to 65.degree. C. to melt the agarose and then cooled to
37.degree. C. for 5 minutes. An equal volume of phenol was added
and the sample was phenol extracted three times (see PHENOL
EXTRACTION). The DNA was then ethanol precipitated and the pellet
resuspended at a concentration of 3-6 fmole DNA/microliter.
LIGATION. DNA was joined together by the action of the enzyme T4
DNA ligase (BRL). Ligation reactions contained 10 fmoles DNA, 20 mM
Tris pH 7.5, 10 mM MgCl.sub.2, 10 mM dithiothreitol (DTT), 200
micromolar ATP, and 20 units T4 DNA ligase in 10 microliters final
reaction volume. The ligation was allowed to proceed for 3-16 hours
at 15.degree. C. Typically DNA fragments to be ligated together
were added at an equal molar ratio. Typically two different DNA
fragments were joined during ligation, but joining of three or four
different DNAs at once was also possible.
RESTRICTION MAPPING OF DNA. Restriction mapping of DNA was
performed as detailed in Maniatis et al. (1). Once it was cloned,
the DNA was digested with a number of different restriction enzymes
and the DNAs were analyzed on agarose gels and the sizes of the
resulting fragments were measured. A double digest with two
different restriction enzymes was performed on the same DNA sample
to aid in the interpretation of the maps. Another approach used was
to cut the DNA with a restriction enzyme that has a single unique
site in the DNA, label the end of the DNA with .sup.32 P using T4
DNA kinase or Klenow DNA polymerase (see POLYMERASE FILL-IN
REACTION) and then cut the DNA with other restriction enzymes at
low temperature or for short times so that only partial digestion
occurred. The subsequent analysis of the partial digestion
fragments on agarose gels served to order the restriction sites on
the map. All of these mapping procedures are well understood by
those skilled in the art and are detailed in Maniatis et al. (1).
The most complete restriction maps can only be composed once the
DNA has been sequenced, and the sequence is then analyzed by a
computer searching for all the known restriction enzyme sites. Some
of our maps have been generated from sequence information.
SOUTHERN BLOTTING OF DNA. The general procedure for Southern
blotting was taken from Maniatis et al. (1). DNA was blotted to
nitrocellulose filters (S&S BA85) in 20.times. SSC (1.times.
SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.0), and prehybridized
in hybridization solution consisting of 30% formamide, 1.times.
Denhardt's solution (0.02% polyvinylpyrrolidone (PVP), 0.02% bovine
serum albumin (BSA), 0.02% Ficoll), 6.times. SSC, 50 mM NaH.sub.2
PO.sub.4, pH 6.8, 200 micrograms/ml salmon sperm DNA for 4-24 hours
at 55 .degree. C. Labeled probe DNA was added that had been
labelled by nick translation using a kit from Bethesda Research
Laboratories (BRL) and one .sup.32 P-labeled nucleotide. The probe
DNA was separated from the unincorporated nucleotides by NACS
column (BRL) or on a Sephadex G50 column (Pharmacia). After
overnight hybridization at 55.degree. C., the filter was washed
once with 2X SSC at room temperature followed by two washes with
0.1.times. SSC, 0.1% sodium dodecyl sulfate (SDS) for 30 minutes at
55.degree. C. The filter was dried and autoradiographed.
DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS. The method is
based upon the calcium phosphate DNA precipitation procedure of
Graham and Van der Eb (34) with the following modifications. For
transfection into animal cells, 0.1-0.2 micrograms of plasmid DNA
containing the foreign DNA flanked by appropriate herepesvirus
cloned sequences (the homovector) were mixed with 0.3 micrograms of
intact DNA. Both DNAs were stored either in H.sub.2 O or 0.01 M
Tris pH 7.5, 1 mM EDTA and the final volume should be less than
0.25 ml. To the mixture was added an equal volume of 2.times. HEPES
buffered saline (10 g N-2-hydroxyethyl piperazine
N'-2-ethanesulfonic acid (HEPES), 16 g NaCl, 0.74 g KCl, , 0.25 g
Na.sub.2 HPO.sub.4. 2H.sub.2 O, 2 g dextrose per liter H.sub.2 O
and buffered with NaOH to pH 7.4). The mixture was then diluted to
0.5 ml by the addition of the appropriate volume of 1X HEPES
buffered saline (prepared by diluting the above solution 1:1 with
H.sub.2 O). After mixing, 35 microliters of 2.2 M CaCl.sub.2 were
added to the DNA mixture and mixed. The mixture was incubated at
room temperature for 30 minutes. Medium was removed from an 80%
confluent monolayer of rabbit skin cells, Vero cells, or CEF cells
growing in a 25 cm.sup.2 flask, and the DNA mixture was added to
the flask and distributed over the cells. After a 30 minute
incubation at room temperature, 5 mls of complete DME medium plus
10% fetal bovine serum were added. The cells were incubated for 5
hours at 37.degree. C. in a humidified incubator containing 5%
CO.sub.2 in air. The medium was changed at 5 hours either with or
without a glycerol shock. When used, the glycerol shock consisted
of removing the medium and adding DME containing 20% glycerol for 3
minutes at room temperature, followed by a wash with 10% glycerol
in DME, and a wash in 5% glycerol in DME, followed by the addition
of fresh complete DME medium plus 10% fetal bovine serum. The cells
were incubated at 37.degree. C. as above for 3-4 days until
cytopathic effect from the virus was 50-100%. Virus was harvested
as described above for the preparation of virus stocks. This stock
was referred to as a transfection stock and it was subsequently
screened for recombinant virus either with or without a selection
mechanism to enrich for recombinant plaques as described below.
DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSES.
Rather than using homovectors and relying upon homologous
recombination to generate recombinant virus, the technique of
direct ligation was developed to insert foreign genes into
herpesviruses. In this instance, the cloned foreign gene did not
require flanking herpesvirus DNA sequences but only required that
it have restriction sites available to cut out the foreign gene
fragment from the plasmid vector. A compatible restriction enzyme
was used to cut the herpesvirus DNA. A requirement of the technique
was that the restriction enzyme used to cut the herepesvirus DNA
must cut at a limited number of sites, preferably less than 3
sites. The herpesvirus DNA was mixed with a 30-fold molar excess of
plasmid DNA, and the mixture was cut with the appropriate
restriction enzyme. The DNA mixture was phenol extracted and
ethanol precipitated to remove restriction enzymes, and ligated
together according to the ligation procedure detailed above. The
ligated DNA mixture was then phenol extracted, ethanol
precipitated, and resuspended in 298 microliters 0.01M Tris pH 7.5,
1 mM EDTA. Forty-two microliters of 2M CaCl.sub.2 were added,
followed by an equal volume of 1.times. HEPES buffered saline (see
above), and the sample was used to transfect animal cells as
described above.
The virus in the transfection stock was then screened for foreign
DNA inserts as described below. The advantage of the direct
ligation technique was that it required less construction of
sub-clones in the plasmid state, and that the recombinant virus was
present in the transfection stock at a much higher frequency than
with homologous recombination.
HAT SELECTION OF RECOMBINANT HERPESVIRUS EXPRESSING THYMIDINE
KINASE. Deletion mutants of herpesviruses which suffered deletions
in the thymidine kinase (TK) gene were constructed. These PRV
strains have been designated S-PRV-002 and S-PRV-003 and have been
deposited with the ATCC under Accession No. VR 2107 and VR 2108
respectively. These TK minus (TK-) viruses have been used as
recipients for the insertion of the foreign herpes simplex type 1
(HSV-1) TK gene. One HSV-1 TK gene that we have used contains the
HSV-1 ICP4 promoter and was from B. Roizman (16). It was sub-cloned
to lie between two flanking regions of PRV DNA, for example by
insertion of the TK gene into PRV BamHI #5 fragment between XbaI
and HpaI sites. The plasmid construct was then transfected with the
PRV TK- DNA to yield recombinant virus. The transfection stock was
enriched for TK-containing virus by the HAT selection procedure
described in (37). The transfection stock was used to infect
monolayers of 143 TK- cells in 60 mm culture dishes that had been
preincubated in HAT medium for 16 hours at 37.degree. C. (HAT
medium: medium 199 containing 2 mM glutamine, 100 units/ml
penicillin, 100 units/ml streptomycin, 10% fetal bovine serum.
5.times.10.sup.5 M hypoxanthine, 10.sup.-5 M thymidine,
5.times.10.sup.-6 M aminopterin). Samples of the transfection stock
virus were infected into the 143 TK- cells using 10.sup.-3 to
10.sup.-7 dilutions of virus. After one or two days at 37.degree.
C., the dishes inoculated with the highest dilution of virus and
still showing virus plaques were harvested for virus stocks, and
the selection was repeated a second time. The virus stock harvested
from the second HAT selection was used in a plaque assay and
individual plaques were picked and tested for foreign DNA inserts
as described below.
BROMODEOXYURIDINE SELECTION OF RECOMBINANT HERPESVIRUS. In order to
insert a foreign gene in place of a TK gene already present in the
herpesvirus genome, the foreign gene was cloned in plasmids so that
it contained the same flanking homology regions as the TK genes.
These flanking regions could be part of the TK gene itself, or
parts of the herpesvirus that flank the TK gene. In either case,
the plasmid DNA containing the foreign gene was transfected with
intact herpesvirus genomic DNA containing the HSV-1 TK gene. The
transfection stock of recombinant virus was grown for two
selections in 143 TK- cells in the presence of 40 micrograms/ml
bromodeoxyuridine (BUDR, Sigma) in complete DME medium plus 10%
fetal bovine serum. The drug BUDR is an analogue of thymidine that
is recognized by the viral enzyme thymidine kinase (TK) and is
ultimately incorporated into DNA. When incorporated into the DNA,
BUDR is mutagenic and lethal and thus selects against viruses that
have an active TK gene. By this selection method, viruses that had
exchanged their TK gene for a foreign gene by homologous
recombination were enriched in the population. Screening for the
recombinant viruses was then performed by one of the techniques
detailed below.
HYBRIDIZATION SCREEN FOR RECOMBINANT HERPESVIRUS. One procedure
used is described in (38). The technique involved doing a plaque
assay on PRV under agarose, removing the agarose once plaques had
formed, and lifting the cell monolayer from the dish onto a
nitrocellulose membrane filter. The filter was then processed
through the Southern procedure for DNA hybridization as detailed
above. The DNA probe used in the procedure was made from the
foreign gene that had been inserted into the virus. Thus plaques
that contain the foreign gene were identified, and they were picked
from the agarose overlay that had been saved.
BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. When the foreign gene
encoded the enzyme beta-galactosidase, the plaques that contained
the gene were visualized more easily. The chemical Bluogal.RTM.
(BRL) was incorporated at the level of 200-300 micrograms/ml into
the agarose overlay during the plaque assay, and the plaques that
expressed active beta -galactosidase turned blue. The blue plaques
were then picked and purified by further blue plaque isolations.
Other foreign genes were inserted by homologous recombination such
that they replaced the beta-galactosidase gene; in this instance
non-blue plaques were picked for purification of the recombinant
virus.
ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS. A third method for
screening the recombinant virus stock was to look directly for the
expression of the foreign gene with antibodies. Herpesvirus plaques
were spotted and picked by inserting a toothpick through the
agarose above the plaque and scraping the plaque area on the dish.
Viruses were then rinsed from the toothpick by inserting the
toothpick into a well of a 96-well microtiter dish (Falcon
Plastics) containing a confluent monolayer of tissue culture cells
that had been washed 3 times in DME medium without serum. It was
important for the virus to grow without serum at this stage to
allow the immunological procedure to work. After cytopathic effect
was complete, the plates were put at -70.degree. C. to freeze and
lyse the cells. The medium was thawed, and the freeze/thaw
procedure was repeated a second time. Then 50-100 microliters of
medium were removed from each well and filtered under vacuum
through a nitrocellulose membrane (S&S BA85) using a
DotBlot.RTM. apparatus (BRL). The filter blots were soaked in a
blocking solution of 0.01M Tris pH 7.5, 0.1M NaCl, 3% bovine serum
albumin at room temperature for two hours with shaking. The filter
blots were then placed in a sealable bag (Sears Seal-A-Meal or
equivalent), and 10 mls of the blocking solution that contained 10
microliters of antibody specific for the foreign protein were
added. After overnight incubation at room temperature with shaking,
the blot was washed 3 times with 100 mls 0.01M Tris, pH 7.5, 0.1M
NaCl, 0.05% Tween 20 detergent (Sigma). The blot was put in another
sealable bag and 10 mls blocking solution containing 10.sup.6
counts per minute of .sup.125 I-protein A (New England Nuclear)
were added. After allowing the protein A to bind to the antibody
for 2 hours at room temperature with shaking, the blot was washed
as above, dried, and overlayed with an x-ray film and an
intensifying screen (Dupont) and autoradiographed for 1-3 days at
-70.degree. C. The film was developed by standard procedures. Virus
from the positive wells which contained the recominant virus was
further purified.
WESTERN BLOTTING PROCEDURE. Samples of cell lysates, positive
controls and protein standards were run on a polyacrylamide gel
according to the procedure of Laemmli (44). After electrophoresis,
the gel was soaked in a transfer buffer (0.025M Tris base, 0.192M
glycine, 20% methanol) plus 0.1% SDS for 20 minutes. The stacking
gel portion was removed and the separation gel was placed onto
Whatman 3 mm paper. A matching-sized piece of nitrocellulose filter
was prewet in the transfer buffer and placed onto the
polyacrylamide gel to cover the gel completely and make intimate
contact. A prewet piece of Whatman 3 mm paper was placed on top of
the nitrocellulose filter to create a "sandwich", and the sandwich
was placed into an electrophoretic transfer device (Biorad). The
sandwich was completely submersed in transfer buffer. The
electrophoretic transfer was carried out for 3 hours at 250
milliamps. After transfer, the nitrocellulose filter was removed
from the assembly and placed in a dish containing 50 mls of
blocking buffer (50 mg/ml bovine serum albumin, 10 mM magnesium
chloride, 100 mM potassium chloride, 1 mM calcium chloride, 10 mM
imidazole pH 7.0, 0.3% Tween-20, 0.02% sodium azide). The
nitrocellulose blot was incubated for 1-2 hours in the blocking
buffer at room temperature on a shaker. The blot was then placed in
a sealable bag containing 15 mls of the blocking buffer plus the
specific antiserum as a probe and incubated overnight at 37.degree.
C. on a shaker. The blot was then removed from the probe solution
and rinsed with 5-6 changes of phosphate buffered saline over a
period of 1 hour. The phosphate buffered saline was removed and 50
mls of blocking buffer containing 5.times.105 cpm of .sup.125 I
labeled protein A (Amersham) were added. The blot was incubated for
1 hour with the labeled protein A solution, the labeled protein A
solution was removed and the blot was rinsed with 5-6 changes of
phosphate buffered saline solution containing 0.3% Tween-20. The
blot was air dried and autoradiographed overnight with an
intensifying screen.
METHOD FOR cDNA CLONING SWINE ROTAVIRUS gp38 GENE Virus Growth. The
OSU strain of porcine rotavirus (ATCC VR-892) was propagated on
MA-104 cells (Rhesus monkey kidney cells from MA Bioproducts).
Confluent monolayers were infected at a multiplicity of infection
of greater than 10 in DMEM containing 5 micrograms/ml trypsin.
Cells were incubated with the virus for 48 hours or until a
cytopathic effect was obtained. Media and cell debris were
collected and centrifuged at 10,000.times. g for 20 minutes at
4.degree. C. The supernatant containing the rotavirus was then
centrifuged at 10,000.times. g in a preparative Beckman Ti45 rotor
at 4.degree. C. Virus pellets were resuspended in SM medium (50 mM
Tris-HCl pH 7.5, 100 mM KCL, 10 mM MgCl.sub.2) and homogenized
lightly in a Dounce-type homogenizer. The resuspended virus was
centrifuged at 10,000.times. g for 10 minutes then loaded onto
25-50% CsCl gradients in SM buffer. Gradients were centrifuged at
100,000.times. g for 4 hours at 20.degree. C. The two blue-white
bands representing intact virions and cores of rotavirus were
collected, diluted, and the CsCl gradient procedure was repeated a
second time. Virus obtained from the second gradient was dialyzed
overnight against SM buffer at 4.degree. C.
Viral RNA Isolation. Dialyzed swine rotavirus was twice extracted
with an equal volume of SDS/phenol then twice more with chloroform:
isoamylalcohol (24:1). The double stranded RNA was precipitated
with ethanol in the presence of 0.2M sodium acetate, centrifuged
and resuspended in water. The yield was typically 100 micrograms
from 1,000 cm.sup.2 of infected cells.
Synthesis and Cloning of gp38 cDNA. 160 micrograms of
double-stranded swine rotavirus RNA obtained from the above
procedure was mixed with one microgram each of two synthetic oligo
nucleotide primers in a volume of 160 microliters (sequences of
primers were: 5'-GGGAATTCTGCAGGTCACATCATACAATTCTAATCTAAG-3' and
5'-GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCCGTTTGGCTA-3') derived from the
published sequence of bovine rotavirus (24). The RNA-primer mixture
was boiled for 3 minutes in a water bath then chilled on ice.
Additions of 25 microliters of 1M Tris-HCl pH 8.3, 35 microliters
of 1M KC1, 10 microliters of 0.25M MgCl.sub.2, 7 microliters of
0.7M 2-mercaptoethanol, 7 microliters of 20 mM dNTP's and 6
microliters of reverse transcriptase (100 units) were made
sequentially. The reaction was incubated at 42 .degree. C. for 1.5
hours then 10 microliters of 0.5M EDTA pH 8.0 was added and the
solution was extracted once with chloroform: phenol (1:1). The
aqueous layer was removed and to it 250 microliters of 4M ammonium
acetate and 1.0 ml of 95% ethanol was added, the mixture was frozen
in dry ice and centrifuged in the cold. The resulting pellet was
resuspended in 100 microliters of 10 mM Tris-HCl pH 7.5 and the
ammonium acetate precipitation procedure was repeated. The pellet
was resuspended in 100 microliters of 0.3M KOH and incubated at
room temperature overnight then at 37.degree. C. for 2 hours. The
solution was brought to neutral pH by addition of 10 microliters of
3.0M HCl and 25 microliters of 1.0M Tris-HCl pH 7.5. The resulting
single-stranded cDNA was then precipitated two times by the above
described ammonium acetate-ethanol procedure. The pellet obtained
was resuspended in 50 microliters of 10 mM Tris-HCl pH 7.5, 100 mM
NaCl, 1 mM EDTA, boiled in a water bath for 2 minutes then
incubated at 59.degree. C. for 16 hours. The solution was
lyophilized to a volume of 15 microliters and the resulting
double-stranded cDNA was run on a 1.0% agarose gel (Sigma agarose
Type II). The ethidium bromide stained DNA migrating at 1,000-1,100
base pair length was excised from the gel and electroeluted in a
CBS electroeluter device. The solution was lyophilized, and the
cDNA was resuspended in 25 microliters of water. To this solution
was added 2 microliters of 1.0M Tris-HCl pH 7.5, 2 microliters of
1M KCl, 1 microliter of 0.25M MgCl.sub.2, 1 microliter of 20 mM
dNTP's and 5 units of E. coli DNA polymerase I. The reaction was
incubated at room temperature for 15 minutes, then
chloroform/phenol extracted and ammonium acetate-ethanol
precipitated as described above. The resulting cDNA was tailed with
dCTP using terminal deoxynucleotide transferase (BRL buffer and
enzyme used). The reaction was stopped with 2 microliters of 0.5M
EDTA, chloroform/phenol extracted and precipitated with sodium
acetate in the presence of 10 micrograms of carrier tRNA. The
resuspended cDNA was mixed with 200 ng of dGMP-tailed Pst I cut
pBR322 (BRL catalog #5355SA) in 200 microliters of 10 mM Tris-HCl
pH 7.5, 100 mM NaCl, 1 mM EDTA, heated to 65.degree. C. for 5
minutes then 57.degree. C. for 2 hours. The annealed cDNA-vector
pBR322 was transformed onto E. coli DH-1 cells prepared for high
efficiency transformation. Colonies that showed sensitivity to
ampicillin and tetracycline resistance were grown and DNA was
prepared and cut with Pst I to determine the size of the cDNA
insert. Several clones having Pst I inserts of 1,050-1,100 base
pairs were analyzed and found to have identical restriction enzyme
digest patterns. The largest clone was designated pSY565 and has
been deposited with the ATCC under accession number 53,340. For one
of these clones, the 1,100 base pair Pst I insert was subcloned
into a M13 phage sequencing vector. The entire DNA sequence of this
clone was determined and is shown in FIG. 10A and 10B. The location
of the gp38 open reading frame was determined from the amino acid
homology to human and bovine sequences already published (24).
METHOD FOR cDNA CLONING BOVINE ROTAVIRUS gp38 GENE Virus Growth.
The Calf Nebraska strain of bovine rotavirus (USDA) was propagated
on MA-104 cells (Rhesus monkey kidney cells from MA Bioproducts).
Confluent monolayers were infected at a multiplicity of infection
of greater than 10 in DMEM containing 5 micrograms/ml trypsin.
Cells were incubated with virus for 48 hours or until a cytopathic
effect was obtained. Media and cell debris were collected and
centrifuged at 10,000.times. g for 20 minutes at 40.degree. C. The
supernatant containing the rotavirus was then centrifuged at
10,000.times. g in a preparative Beckman Ti45 rotor at 4.degree. C.
Virus pellets were resuspended in SM medium (50 mM Tris-HCl pH 7.5,
100 mM KCL, 10 mM MgCl.sub.2) and homogenized lightly in a
Dounce-type homogenizer. The resuspended virus was centrifuged at
10,000.times. g for 10 minutes then loaded onto 25-50% CsCl
gradients in SM buffer. Gradients were centrifuged at
100,000.times. g for 4 hours at 20.degree. C. The two blue-white
bands representing intact virions and cores of rotavirus were
collected, diluted, and the CsCl gradient procedure was repeated a
second time. Virus obtained from the second gradient was dialyzed
overnight against SM buffer at 4.degree. C. Viral RNA Isolation.
Dialyzed bovine rotavirus was twice extracted with an equal volume
of SDS/phenol then twice more with chloroform: isoamylalcohol
(24:1). The double stranded RNA was precipitated with ethanol in
the presence of 0.2M sodium acetate, centrifuged and resuspended in
water. The yield was typically 100 micrograms from 1,000 cm.sup.2
of infected cells.
Synthesis and Cloning of gp38 cDNA. 160 micrograms of
double-stranded bovine rotavirus RNA obtained from the above
procedure was mixed with one microgram each of two synthetic oligo
nucleotide primers in a volume of 160 microliters (sequences of
primers were: 5'-GGGAATTCTGCAGGTCACATCATACAATTCTAATCTAAG-3' and
5'-GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCOGTTTGGCTA-3') derived from the
published sequence of bovine rotavirus (24). The RNA-primer mixture
was boiled for 3 minutes in a water bath then chilled on ice.
Additions of 25 microliters of 1M Tris-HCl pH 8.3, 35 microliters
of 1M KC1, 10 microliters of 0.25M MgCl.sub.2, 7 microliters of
0.7M 2-mercaptoethanol, 7 microliters of 20 mM dNTP's, and 6
microliters of reverse transcriptase (100 units) were made
sequentially. The reaction was incubated at 42.degree. C. for 1.5
hours then 10 microliters of 0.5M EDTA pH 8.0 was added and the
solution was extracted once with chloroform: phenol (1:1). The
aqueous layer was removed and to it 250 microliters of 4M ammonium
acetate and 1.0 ml of 95% ethanol was added, the mixture was frozen
in dry ice and centrifuged in the cold. The resulting pellet was
resuspended in 100 microliters of 10 mM Tris-HCl pH 7.5 and the
ammonium acetate precipitation procedure was repeated. The pellet
was resuspended in 100 microliters of 0.3M KOH and incubated at
room temperature overnight then at 37.degree. C. for 2 hours. The
solution was brought to neutral pH by addition of 10 microliters of
3.0M HCl and 25 microliters of 1.0M Tris-HCl pH 7.5.
The resulting single-stranded CDNA was then precipitated two times
by the above described ammonium acetate-ethanol procedure. The
pellet obtained was resuspended in 50 microliters of 10 mM Tris-HCl
pH 7.5, 100 mM NaCl, 1 mM EDTA, boiled in a water bath for 2
minutes then incubated at 59.degree. C. for 16 hours. The solution
was lyophilized to a volume of 15 microliters and the resulting
double-stranded CDNA was run on a 1.0% agarose gel (Sigma agarose
Type II). The ethidium bromide stained DNA migrating at 1,000-1,100
base pair length was excised from the gel and electroeluted in a
CBS electroeluter device. The solution was lyophilized, and the
CDNA was resuspended in 25 microliters of water. To this solution
was added 2 microliters of 1.0M Tris-HCl pH 7.5, 2 microliters of
1M KCl, 1 microliter of 0.25M MgCl.sub.2, 1 microliter of 20 mM
dNTP's, and 5 units of E. coli DNA polymerase I. The reaction was
incubated at room temperature for 15 minutes, then
chloroform/phenol extracted and ammonium acetate-ethanol
precipitated as described above. The resulting CDNA was tailed with
dCTP using terminal deoxynucleotide transferase (BRL buffer and
enzyme used). The reaction was stopped with 2 microliters of 0.5M
EDTA, chloroform/phenol extracted and precipitated with sodium
acetate in the presence of 10 micrograms of carrier tRNA. The
resuspended cDNA was mixed with 200 ng of dGMP-tailed Pst I cut
pBR322 (BRL catalog #5355SA) in 200 microliters of 10 mM Tris-HCl
pH 7.5, 100 mM NaCl, 1 mM EDTA, heated to 65.degree. C. for 5
minutes then 57.degree. C. for 2 hours. The annealed cDNA-vector
pBR322 was transformed onto E. coli DH-1 cells prepared for high
efficiency transformation. Colonies that showed sensitivity to
ampicillin and tetracycline resistance were grown and DNA was
prepared and cut with Pst I to determine the size of the cDNA
insert. Several clones having Pst I inserts of 1,050-1,100 base
pairs were analyzed and found to have identical restriction enzyme
digest patterns. For one of these clones, the 1,100 base pair Pst I
insert was subcloned into a M13 phage sequencing vector. Part of
the DNA sequence of this clone was determined and was found to be
identical to the published sequence (24).
SELECTION OF G418 RESISTANT HERPESVIRUS. The antibiotic G418
(GIBCO) has a wide range of inhibitory activity on protein
synthesis. The recombinant virus, however, expressed the
aminoglycoside 3'-phosphotransferase, encoded by the NEO gene, upon
acquiring the foreign gene and became resistant to G418. The
transfection stocks of recombinant viruses were grown on MDBK (for
IBR virus), Vero (for PRV) or QT35 (for HVT) cells in the presence
of 500 micrograms/ml G418 in complete DME medium plus 1% fetal
bovine serum. After one or two days at 37.degree. C., plaques from
the dishes inoculated with the highest dilution of virus were
picked for virus stocks. The selection was repeated a second or
third time. The virus stocks generated from the G418 selection were
tested for NEO gene insertion by the SOUTHERN BLOTTING OF DNA
hybridization procedure described above.
VACCINATION STUDIES IN SWINE. Weaned pigs (4-6 weeks old) and
pregnant sows were obtained from swine herds known to be free of
pseudorabies disease. Susceptibility of the test animals to
pseudorabies was further verified by testing the pig serum for
absence of neutralizing antibodies to pseudorabies virus (PRV). The
weaned pigs and 3-to-4 day old piglets were inoculated
intramuscularly with 1 ml of virus fluid containing about 10.sup.4
to 10.sup.6 infectious units (TCID.sub.50) Animals were observed
each day after vaccination for adverse reactions (clinical signs of
PRV disease) and body temperatures were recorded. Samples of
tonsillar secretions were obtained and cultured to determined if
the vaccine virus was capable of shedding and spreading to other
animals. Immunity was determined by measuring PRV serum antibody
levels at weekly intervals and in some cases, by challenging the
vaccinated pigs with virulent virus. In the latter case, the
vaccinated animals and a group of non-vaccinated pigs were
inoculated with virulent, Shope strain PRV, using an amount of
virus that caused PRV disease in at least 80% of the unvaccinated
group of pigs. This was done about 28 days after vaccination. The
challenged animals were observed daily for signs of disease and for
increased body temperatures. A necropsy was conducted on animals
that died and selected tissues were examined and cultured for
PRV.
EXAMPLES
Example 1
S-PRV-004
We have created a virus that has a deletion in the junction region
between the unique long DNA and the internal repeat of PRV, and a
deletion in the endogenous PRV thymidine kinase gene in the unique
long region. Into the junction deletion we have cloned the herpes
simplex type 1 (HSV-l) thymidine kinase (TK) gene under the control
of the ICP4 promoter. This virus is designated S-PRV-004.
To create this virus, we first cloned the SalI #1 fragment of PRV.
PRV DNA was prepared and then cut with SalI restriction enzyme. The
cut DNA was electrophoresed on an agarose gel and the largest SailI
band (15 kb) was purified from the gel (see PHENOL EXTRACTION OF
DNA FROM AGAROSE). The purified DNA was ligated into the plasmid
pSP64 (see LIGATION) and the DNA mixture was used to transform E.
coli HB101 according to Maniatis et al. (1). The SalI #1 clone was
mapped for restriction sites.
The homologous recombination procedure was used to create S-PRV-004
(see FIG. 2). The exact position of the junction region was
determined by sequencing the DNA from SalI #1 fragment. It was
found that the junction region was positioned between two StuI
sites (FIG. 2A). Two fragments of DNA from the SalI clone were used
to create the homology vector for recombination. One was a fragment
from BamHI #8' from StuI to BamHI and the other was from BamHI #8
from BamHI to StuI (see FIGS. 1B and 2A). These fragments were
cloned into the BamHI site of pSP64. This plasmid was cut with
StuI, and a 3.8 kb PvuII fragment, obtained from B. Roizman (16),
The University of Chicago, and containing the ICP4 promoter on the
BamHI-N fragment and the HSV-1 TK gene on the BamHI-Q fragment,
fused at the BamHI/BglII sites, was ligated into the StuI site. The
net result from this series of clonings was a plasmid which had
suffered a deletion of 3kb from between the StuI sites, and into
which 3.8kb of the foreign TK gene had been incorporated (see FIG.
2B). The TK gene was thus flanked by PRV DNA sequences to allow for
insertion of the foreign gene into the PRV genome by homologous
recombination. The plasmid DNA was tranfected into rabbit skin
cells along with the intact PRV DNA from S-PRV-003, which is a
pseudorabies virus that has a deletion in the endogenous TK gene.
The transfection stock of virus was selected in HAT medium and the
virus was identified and selected by analysis of the restriction
pattern of DNA isolated from the infected cells.
S-PRV-004 contained the HSV-1 TK gene and was expressing this gene
as demonstrated by the incorporation of 14C-thymidine in a plaque
assay described in Tenser et al. (42) and by direct analysis of TK
activity in infected cell extracts, following the procedure of
Cheng et al. (43). The location of this gene in the genome of PRV
is shown in FIG. 2C.
Six weaning age pigs were vaccinated with 10.sup.5.0 infectious
units of S-PRV-004 and challenged with virulent PRV 28 days later,
according to the VACCINATION STUDIES IN SWINE procedure. The
vaccinated pigs remained healthy following vaccination and
developed serum neutralizing antibody against PRV (see Table I
below). Vaccine virus was not recovered from nasal or tonsillar
secretions. After exposure to virulent PRV, 83% of the vaccinated
swine were protected against PRV disease.
TABLE I
__________________________________________________________________________
RESPONSES OF WEANED PIGS VACCINATED WITH S-PRV-004 AND CHALLENGED
WITH VIRULENT PRV Post-Vaccination Post-Challenge Antibody Antibody
Antigen Pig Day Day Day Clinical Virus Day Day Clinical Virus Level
No. 14 21 28 Signs Isolation 7 14 Signs.sup.a Isolation
__________________________________________________________________________
10.sup.5.0 1 32 32 16 None None >64 >64 F Swabs 2 16 32 8
None None >64 >64 F Swabs 3 8 16 4 None None >64 >64 F
Swabs 4 4 16 8 None None >64 >64 F,C Swabs 5 16 16 8 None
None >64 >64 F Swabs 6 8 8 4 None None >64 >64 F Swabs
__________________________________________________________________________
.sup.a Key to clinical signs: C = CNS, F = Febrile
Example 2
S-PRV-005
S-PRV-005 is a pseudorabies virus that has a deletion in the repeat
region and in the endogenous PRV TK gene in the long unique region,
and has an insertion of the HSV-1 TK gene under the control of the
ICP4 promoter incorporated into both copies of the repeat region
between the XbaI site and the HpaI site in the BamHI #5 fragment
(See FIG. 3A-3C).
To create this virus, we first obtained a clone of BamHI #5
fragment from PRV (FIG. 1B). The BamHI #5 fragment was cloned into
the plasmid pACYC184 at the BamHI site (see LIGATION above). A map
of the BamHI #5 fragment is shown in FIG. 3A.
The plasmid containing the BaMHI #5 fragment was cut with XbaI and
HpaI and the linearized plasmid was purified (see PHENOL EXTRACTION
OF DNA FROM AGAROSE). The 3.8kb PvuII fragment described in Example
1 and containing the TK gene and ICP4 promoter was likewise
purified. The XbaI site was filled to yield a blunt end (see
POLYMERASE FILL-IN REACTION), and the two DNAs were mixed and
ligated together. The resulting plasmid that had incorporated the
TK gene in the XbaI-HpaI deletion was selected and analyzed by
restriction mapping (FIG. 3B).
The plasmid containing the TK gene flanked by PRV Bam HI #5
sequences was used to transfect rabbit skin cells along with
purified DNA from S-PRV-003, a pseudorabies virus that had a
deletion in the endogenous TK gene. The resulting recombinant PRV
that had incorporated the HSV-1 TK gene into the deletion in the
repeats was screened and purified from the transfection stock by
the HYBRIDIZATION SCREEN FOR RECOMBINANT HERPESVIRUS procedure
without any prior selection.
S-PRV-005 recombinant PRV was shown to express the HSV-1 TK gene by
incorporation of .sup.14 C-thymidine in a plaque assay (42), by
analysis of the TK activity in infected cell lysates (43), and by
immunodetection of the HSV-1 TK protein according to the ANTIBODY
SCREEN FOR RECOMBINANT HERPESVIRUS procedure outlined above. The
location of this gene in the genome of PRV is shown in FIG. 3C.
Example 3
S-PRV-010
S-PRV-010 is a pseudorabies virus that has a deletion in the PRV TK
gene in the long unique region, a deletion in the repeat region,
and the insertion of the E. coli beta-galactosidase gene (lacZ
gene) incorporated into both copies of the repeats at the XbaI site
in BamHI #5 fragment (see FIG. 5A). The beta-galactosidase gene was
constructed to be expressed using the HSV-1 TK gene promoter which
we have shown in this construct to be active in PRV.
The method used to insert the beta-galactosidase gene into
S-PRV-010 was direct ligation (see DIRECT LIGATION PROCEDURE FOR
GENERATING RECOMBINANT HERPESVIRUS). The beta-galactosidase gene
was on plasmid pJF751, obtained from Jim Hoch, Scripps Clinic and
Research Foundation. This gene is truncated at the 5' end with a
BamHI site that has removed the AGT initiation codon, and the AvaI
site in pBR322 was used at the other end (see FIG. 4A). The HSV-1
TK promoter (FIG. 4B) was taken from the McKnight TK gene as an
RsaI fragment, gel purified, and ligated to a synthetic piece of
DNA which contained a BamHI site within the sequence CGGATCCG (FIG.
4C). After digestion with BamHI, the fragment was cloned into the
BamHI site at the start of the beta-galactosidase gene (FIG. 4D).
The plasmid was constructed with the E. coli plasmids pSP64 and
pSP65 such that XbaI sites from the polylinkers could be used to
excise the entire construct from the plasmid. The ligation mixture
was used to transfect E. coli HB101 according to published
procedures (Maniatis et al. (1)). This construct was planned such
that the first three amino acids of the protein were from the HSV-1
TK gene, the next three were from the synthetic linker, and the
rest were from the beta-galactosidase gene. The gene contained the
following sequence at the fusion between TK and lacZ: ##STR1##
A pseudorabies virus construct designated S-PRV-002 which has a
deletion in the PRV TK gene in the unique long region and a
deletion in the repeat region was used as the recipient for the
beta-galactosidase gene. Intact S-PRV-002 DNA was mixed with a
30-fold molar excess of plasmid DNA containing the
beta-galactosidase gene under the control of the HSV-1 TK promoter,
and this mixture was digested with XbaI restriction enzyme. The
ligated DNA was used to transfect animal cells, and the
transfection stock was analyzed for recombinant PRV. First, PRV DNA
was prepared from cells infected with the transfection stock virus
and this DNA was cut with restriction enzymes and analyzed on an
agarose gel. This analysis showed that the recombinant virus was
present as the major species in the transfection stock, and it was
subsequently purified from other virus species by plaque assay
coupled with the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS.
Because beta-galactosidase reacted with the drug Bluogal.RTM. to
yield a product with blue color, it was possible to plaque purify
the recombinant by picking blue plaques.
The final result of the purification was the recombinant PRV
designated S-PRV-010. It was shown to express the enzyme
beta-galactosidase by the formation of blue plaques as noted above,
and by the detection of the enzyme in infected cell extracts using
the substrate O-nitrophenyl-beta-D-galactopyranoside (Sigma)
following the procedure of Norton and Coffin (35). The location of
this gene in the genome of PRV is shown in FIG. 5C.
Previous studies demonstrated that swine vaccinated with S-PRV-002
developed antibody to PRV and were fully protected against clinical
disease following exposure to virulent PRV virus. Animal studies
were conducted with S-PRV-010 to determine the utility of a
recombinant pseudorabies virus as a vaccine against pseudorabies
disease.
A group of weaned pigs and a litter of four-day-old piglets were
vaccinated with S-PRV-010 and challenged three to four weeks later,
according to VACCINATION STUDIES IN SWINE.
Responses of weaned pigs vaccinated with S-PRV-010 are shown in
Table II. Administration of this virus did not cause adverse
reactions in the pigs. The vaccinated animals developed PRV
neutralizing antibody. Two, non-vaccinated control animals (#75 and
#91) placed in contact with the vaccinates did not develop PRV
antibody prior to challenge, indicating the vaccine virus was not
shed from vaccinates. After challenge, all ten vaccinated animals
remained clinically normal and free of PRV disease. In contrast,
the two in-contact control animals and three of five non-vaccinated
control animals developed PRV disease and one of these pigs died of
PRV.
To test further the utility of S-PRV-010 as a vaccine, the virus
was inoculated into 4-day old piglets. The results, presented in
Table III, demonstrated that the virus elicited an antibody
response in vaccinated piglets and did not cause adverse reactions.
The virus apparently was shed from vaccinates, since one (#67) of
two non-vaccinated, in-contact control piglets had developed PRV
antibody by Day 24. After challenge, all vaccinated animals and the
sero-positive in-contact control animal remained free of PRV
disease. By comparison, the three non-vaccinated control pigs and
the second in-contact control pig developed clinical signs of PRV
and died.
The conclusion from that study is that S-PRV-010 given at a dosage
of 10.sup.4.0 or 10.sup.6.0 , elicits a protective response in
vaccinated piglets or weaned pigs capable of preventing infection
by virulent virus.
TABLE II
__________________________________________________________________________
SEROLOGIC AND CLINICAL RESPONSES OF WEANED PIGS FOLLOWING
VACCINATION WITH S-PRV-010 AND CHALLENGE WITH WILD-TYPE PRV
Antibody Titers.sup.a Post- Post- Post-Challenge Vaccine Pig
Vaccination Challenge Clinical GROUP Number Day 0 Day 14 Day 24 Day
7 Day 14 Signs
__________________________________________________________________________
10.sup.6.0 70 <2 64 32 32 64 None Per 71 <2 16 16 16 32 None
Dose 72 <2 64 32 16 64 None 73 <2 64 16 16 64 None 74 <2
16 8 4 4 None 75.sup.b <2 <2 <2 <2 4 Depressed,
Dyspnea, CNS Signs.sup.c 10.sup.4.0 76 <2 64 4 8 32 None Per 77
<2 16 16 64 8 None Dose 78 <2 32 16 32 8 None 79 <2 8 16
64 4 None 80 <2 2 <2 256 16 None 81.sup.b <2 <2 <2
<2 16 Depressed Rhinitis, CNS Signs Con- 82 NT NT <2 <2 8
None Trols 83 NT NT <2 <2 16 None 84 NT NT <2 <2 32 CNS
Signs, Depressed, Dyspnea 85 NT NT <2 <2 64 CNS Signs 86 NT
NT <2 <2 -- CNS Signs, Died
__________________________________________________________________________
.sup.a Determined by RIDEA .sup.b Incontact Controls .sup.c CNS
signs include Ataxia, Incoordination, Circling, Lateral Recumbency
NT: Not Tested
TABLE III
__________________________________________________________________________
SEROLOGIC AND CLINICAL RESPONSES OF 4-DAY-OLD PIGLETS FOLLOWING
VACCINATION WITH S-PRV-010 AND CHALLENGE WITH WILD-TYPE PRV
Antibody Titers.sup.a Post- Post- Post-Challenge Vaccine Pig
Vaccination Challenge Clinical GROUP Number Day 0 Day 14 Day 24 Day
7 Day 14 Signs
__________________________________________________________________________
10.sup.6.0 60 <2 4 16 16 32 None Per 61 <2 64 8 64 8 None
Dose 62 <2 32 2 16 16 None 10.sup.4.0 63 <2 --.sup.b -- -- --
-- Per 64 <2 64 2 32 16 None Dose 65 <2 2 4 32 16 None
In-Contact 66 <2 2 NT --.sup.c -- Comatose, Died Controls 67
<2 <2 8 64 32 None Controls 87 NT NT <2 --.sup.c -- CNS
Signs.sup.d, Died 88 NT NT <2 --.sup.c -- CNS Signs, Died 89 NT
NT <2 --.sup.c -- Died
__________________________________________________________________________
.sup.a Determined by RIDEA .sup.b Died 8 Days Post Vaccination From
Ruptured Stomach .sup.c Died on or Prior to Day 7 PostChallenge
.sup.d CNS Signs include Ataxia, Incoordination, Circling Lateral
Recumbency NT: Not Tested
Example 4
S-PRV-007 is a pseudorabies virus that has a deletion in the PRV TK
gene in the unique long region, a deletion in the repeat region,
and the swine rotavirus glycoprotein 38 gene under the control of
the HSV-1ICP4 promoter inserted into the repeat region.
S-PRV-005 virus described in Example 2 above was further engineered
to contain the rotavirus antigen (see FIGS. 6A-6C) as follows. The
swine rotavirus gp38 gene was cloned into plasmid pBR322 at the
PstI site by procedures previously described herein. The resulting
plasmid was called pSY565 (see FIG. 7). The 1090 bp PstI fragment
containing the gp38 gene was cloned into vector pUC4K at the PstI
site such that it became flanked by BamHI sites in a plasmid called
pSY762.
Plasmid pSY590 has had a complex origin as inferred from the flow
chart. These clonings were routine in nature and are of historical
interest but are not strictly required to practice the invention.
Briefly this history is:
(1) The McKnight TK gene was the HSV-1BamHI Q fragment from HaeIII
at -178 relative to CAP site to BamHI at +2700 which was cloned
between HindIII and BamHI in pBR327.
(2) pSY491..The entire TK coding region from the BglII site at +55
(relative to CAP site) to the BamHI site at +2700 was cloned into
the BamHI site in pSP65 and called pSY491.
(3) pSY481..The polyA signal sequence (pA) on an 800 bp SmaI
fragment from TK was subcloned into the SmaI site in pSP65 and was
called pSY481.
(4) pSY583..The pA 800 bp SmaI fragment from pSY481 was cloned into
the HincII site in pSP65 and called PSY583.
(5) pSY429..The HSV-1 BamHI N fragment was obtained from Dr. B.
Roizman cloned into the BamHI site of pBR322 and was called
pSY429.
(6) pSY584..The 2.2kb fragment of BamHI N from PvuII to BamHI (ref.
16) in pSY420 was subcloned into pSP65 between HincII and BamHI in
the polylinker and was called pSY584.
(7) pSY479..A plasmid was constructed from pSP64 and pSP65 that
contained a fused polylinker sequence. Both plasmids were cut with
PstI in the polylinker and PvuI in the plasmid body. The net effect
of this construct was to create a fusion plasmid called pSP66 which
has a symmetrical polylinker sequence centered on the PstI site.
pSY479 is the name of this plasmid and it also contained a PstI
fragment cloned into the PstI site that is irrelevant for the
manipulations that follow.
Plasmid pSY590 was created from pSY583, pSY584, and pSY479 in a
three fragment ligation of the following elements: the 3kb plasmid
sequences from pSY479 (pSP66) cut with PstI, the 800 bp SmaI pA
fragment cut from the polylinker in pSY583 with PstI and BamHI, and
the 2200bp BamHI N fragment cut from pSY583 with PstI and BamHI.
FIG. 7 shows the final configuration of all of these DNA fragments
in pSY590. There is a single BamHI site in the plasmid between the
promoter in BamHI N and the TK pA signal that was used to insert
the coding region of the gp38 gene.
For the creation of the homology vector used in the formation of
S-PRV-007, the plasmid pSY590 was opened with BamHI, and the 1090
bp gp38 gene was removed from pSY762 by cutting with BamHI, and
these two fragments were ligated together to form pSY596. The
correct orientation of the gp38 gene was confirmed by diagnostic
restriction enzyme digestion utilizing sites with gp38 (see FIG.
10A and 10B).
In pSY596 described above, the gp38 gene resided between two
flanking HSV-1 DNA fragments. These two regions were thus
homologous to similar regions on the HSV-1 TK gene in S-PRV-005,
and these regions were used for the homologous recombination to
create S-PRV-007 (FIG. 6A). The plasmid and S-PRV-005 DNAs were
mixed and used in the DNA TRANSFECTION PROCEDURE FOR GENERATING
RECOMBINANT VIRUS. A virus that had incorporated the rotavirus
antigen in place of the TK gene was selected with BUDR.
Recombinants from the selected virus stock that had incorporated
the rotavirus DNA were screened by the HYBRIDIZATION SCREEN FOR
RECOMBINANT HERPESVIRUS and by analyzing restriction digests of DNA
by the SOUTHERN BLOTTING OF DNA procedure using the rotavirus
cloned gp38 gene as probe.
The final result of this screening was a recombinant PRV called
S-PRV-007 which had the rotavirus gp38 gene incorporated into the
repeat region between the XbaI and HpaI sites in PRV BamHI #5
fragment shown in FIG. 6C. The presence in a host of gp38 expressed
by S-PRV-007 has not yet been detected.
Example 5
S-PRV-012
S-PRV-012 is a pseudorabies virus that has a deletion in the PRV TK
region in the unique long region, a deletion in the repeat region,
and a deletion in the unique short region encoding the PRV
glycoprotein X, called gpX and identified and mapped by Rea et al.
(23). The HSV-1 TK gene under the control of the ICP4 promoter was
inserted in place of the gpX gene.
The following procedure was used to make the deletion of gpX and
the simultaneous insertion of the HSV-1 TK gene. The flanking
regions for homology to PRV were from cloned fragments of BamHI #10
fragment and BamHI #7 fragment extending from NdeI to BamHI (FIG.
8A-8C). The BamHI and NdeI sites were filled in according to the
POLYMERASE FILL-IN REACTION, and the PvuII fragment of HSV-1 DNA
was inserted by LIGATION. This plasmid was transfected with intact
S-PRV-002 DNA according to the DNA TRANSFECTION FOR GENERATING
RECOMBINANT VIRUS procedure. The recombinant virus was selected by
HAT SELECTION OF RECOMBINANT HERPESVIRUS procedure, and screened by
the ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS procedure using
antibodies specific for the HSV-1 protein.
The recombinant virus selected by this procedure was designated
S-PRV-012 and has been deposited with the ATCC under Accession No.
VR-2119 and was shown by RESTRICTION MAPPING OF DNA and SOUTHERN
BLOTTING OF DNA to contain the HSV-1 TK gene inserted in place of
the gpX gene (FIG. 8B). The ANTIBODY SCREEN FOR RECOMBINANT
HERPESVIRUS procedure showed that the virus was expressing the
inserted HSV-1 TK gene. The structure of this virus is shown in
FIG. 8C.
Example 6
S-PRV-013
S-PRV-013 is a pseduorabies virus that has a deletion in the TK
gene in the long unique region, a deletion in the repeat region and
a deletion in the gpX coding region. The gene for E. coli
beta-galactosidase (lacZ gene) was inserted in place of the gpX
gene and is under the control of the endogenous gpX gene
promoter.
The following procedures were used to construct S-PRV-013 by
homologous recombination. The flanking PRV homology regions were
from the cloned BamHI #10 fragment which contained the gpX
promoter, and from the cloned BamHI #7 fragment extending from the
NdeI site to the BamHI site (FIG. 9A). The NdeI site was filled in
according to the POLYMERASE FILL-IN REACTION, and the
beta-galactosidase gene was inserted between the BamHI #10 and
BamHI #7 fragments. This construct positioned the
beta-galactosidase gene behind the gpX promoter and the gpX poly A
signal sequences with a deletion of almost all of the coding
regions of gpX. The plasmid DNA and DNA from S-PRV-002, a PRV
strain with a deletion in both repeat sequences and a deletion in
the thymidine kinase gene, were mixed and transfected according to
the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure.
The recombinant virus was screened and purified from the
transfection stock by the BLUOGAL SCREEN FOR RECOMBINANT
HERPESVIRUS procedure.
The resulting virus from this screen was designated S-PRV-013 and
has been deposited with the ATCC under Accession No. VR 2120. It
contained the beta-galactosidase gene in place of the gpX coding
regions (FIGS. 9B and 9C) as determined by PREPARATION OF
HERPESVIRUS DNA followed by SOUTHERN BLOTTING OF DNA. The
expression of the beta-galactosidase gene was confirmed by the
BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS test, and by the
o-nitrophenyl-galactopyranoside substrate assay (27).
To confirm that the coding region for gpX had been removed from
S-PRV-013, DNA extracted from a stock of purified S-PRV-013 was
digested with BamHI and the fragments were separated on agarose gel
electrophoresis and analyzed by SOUTHERN BLOT HYBRIDIZATION. The
hybridization probe was the BamHI-NDE fragment of pseudorabies
BamHI #7 fragment from the unique short region. This probe fragment
included 90% of the coding sequences of gpX. In this analysis, the
gpX region was shown to be missing from S-PRV-013.
The following experiments indicate that S-PRV-013 may be used as a
vaccine to protect swine against psuedorabies disease. In the first
study, susceptible weaned pigs and four-day old piglets were
vaccinated intramuscularly with S-PRV-013 as follows: 4 of each
group were inoculcated with 10.sup.6 TCID.sub.50 and 4 were
inoculated with 10.sup.4 TCID.sub.50 of virus. The animals were
observed, then challenged as described in VACCINATION STUDIES WITH
SWINE (see Table IV below)
TABLE IV
__________________________________________________________________________
RESPONSES OF 4-DAY-OLD PIGLETS VACCINATED WITH S-PRV-013 AND
CHALLENGED WITH VIRULENT PRV Post-Vaccination Post-Challenge Pig
Vaccine Pig Antibody Clinical Virus Antibody Clinical Group Dose
No. Day 14 Day 21 Day 28 Signs.sup.a Isolation Day 7 Day 14 Signs
__________________________________________________________________________
WEANED 10.sup.6 1 4 2 2 NEG NT.sup.b >64 >64 NEG TCID.sub.50
2 4 2 2 NEG NT >64 >64 NEG 3 2 2 2 NEG NT 64 >64 NEG 4 4 2
4 NEG NT >64 >64 NEG 10.sup.4 5 2 2 2 NEG NT >64 >64
NEG TCID.sub.50 6 <2 <2 2 NEG NT 64 >64 NEG 7 <2 <2
<2 NEG NT 64 >64 NEG 8 <2 <2 <2 NEG NT 64 >64 NEG
PIGLETS 10.sup.6 10 8 16 32 NEG NEG >64 >64 NEG TCID.sub.50
11 --.sup.c -- -- NEG NEG -- -- -- 12 8 NT 64 NEG NEG >64 >64
NEG 13 4 8 32 NEG NEG >64 >64 NEG 10.sup.4 14 4 8 32 NEG NEG
>64 >64 NEG TCID.sub.50 15 8 16 32 NEG NEG >64 >64 S 16
2 2 8 NEG NEG 64 >64 NEG 17 4 16 32 NEG NEG 64 64 NEG Contact 18
<2 <2 <2 NEG NEG 2 16 F,C Control 19 --.sup.d -- -- NEG
NEG -- -- -- Challenge 20 Not Applicable <2 -- F,C,I Control 21
<2 2 F,C 22 <2 2 F,C
__________________________________________________________________________
.sup.a Key to clinical signs: NEG = Negative, C = CNS, D = Death, F
= Febrile, R = Respiratory, S = Scours .sup.b Not tested .sup.c
Sacrificied Day 4 postvaccination .sup.d Sacrificed Day 7
postvaccination; runt doing poorly
Following vaccination, all animals were free of adverse reactions
and all but 2 (weaned pigs) developed serum neutralizing antibody
titers of 1:2 to 1:64. Virus was not recovered from tonsillar swabs
of any pig or from tissues taken from the piglet (#11) sacrificed
on Day 4. One of 2 contact control piglets (#19) was sacrificed 7
days into the experiment because it was a runt and doing poorly.
Tissues from this piglet were negative when cultured for PRV. The
other contact control remained healthy and did not develop PRV
antibody prior to challenge.
After challenge, all vaccinated animals remained clinically normal
and developed secondary antibody responses. The contact control
piglet and the three challenge control pigs all developed typical
central nervous system signs of PRV and one control died following
challenge.
In a second study with S-PRV-013 using larger numbers of animals, 2
litters of susceptible 3-day-old piglets and a group of 15
susceptible weaned pigs were vaccinated with 10.sup.4 TCID.sub.50
of virus, then challenged as described in VACCINATION STUDIES WITH
SWINE (see Tables V and VI below).
TABLE V
__________________________________________________________________________
RESPONSES OF 3-DAY-OLD PIGLETS VACCINATED WITH S-PRV-013 AND
CHALLENGED WITH VIRULENT PRV Post-Vaccination Post-Challenge
Antibody Antibody Pig Day Day Day Day Clinical Virus Day Day
Clinical Virus Group No. 7 14 21 28 Signs.sup.a Isolation 7 14
Signs Isolation
__________________________________________________________________________
LITTER 1 <2 2 4 4 F.sup.b Neg 32 >64 Neg Neg A 2 <2 8 8 16
F Neg 64 >64 Neg Neg VACCINATES 3 <2 8 8 16 F Neg 16 32 Neg
Neg 4 <2 8 16 16 F Neg 32 >64 Neg Neg 6 <2 8 8 16 F Neg 64
>64 Neg Neg Contact 7 <2 <2 <2 <2 Neg Neg 2 2 C,F,R
Neg Control 8 <2 <2 <2 <2 Neg Neg 2 >64 C,F Neg
LITTER 10 <2 8 8 16 F Neg 16 >64 Neg Neg B 11 <2 8 8 16 F
Neg 32 >64 Neg Neg VACCINATES 12 <2 8 32 32 F Neg 32 >64
Neg Neg 13 <2 4 16 32 F Neg 64 >64 Neg Neg 14 <2 8 16 32
Neg Neg 64 >64 Neg Neg 16 <2 4 4 16 F Neg 32 >64 Neg Neg
17 <2 8 8 32 F Neg 64 >64 Neg Neg Contact 18 <2 <2
<2 <2 Neg Neg 2 2 C,F Neg Control CHALLENGE 19 Not <2 Not
<2 2 C,F,R Neg CONTROLS 20 Applicable <2 Applicable <2 2
C,F,R Swab 21 <2 <2 <2 C,F,R Swab 22 <2 <2 <2
C,F,R Swab 23 <2 <2 Died C,D,F,R Swab Tonsil, CNS 24 <2
<2 <2 C,F,R Swab
__________________________________________________________________________
.sup.a Clinical signs: NEG = Negative, C = CNS, D = Death, F =
Febrile, R = Respiratory .sup.b A 1.degree. F. increase in
temperature was observed in day 1 in these vaccinates
TABLE VI
__________________________________________________________________________
RESPONSE OF WEANED PIGS VACCINATED WITH S-PRV-013 AND CHALLENGED
WITH VIRULENT PRV Post-Vaccination Post-Challenge Antibody Antibody
Pig Day Day Day Clinical Virus Day Day Clinical Virus Group No. 0
14 21 Signs.sup.a Isolation 7 14 Signs Isolation
__________________________________________________________________________
VACCINATES 35 <2 <2 4 Neg Neg >64 >64 Neg Neg 36 <2
2 2 Neg Neg >64 >64 Neg Neg 37 <2 2 2 Neg Neg >64
>64 Neg Neg 38 <2 <2 2 Neg Neg >64 >64 Neg Neg 39
<2 2 2 Neg Neg 64 64 Neg Neg 40 <2 2 4 Neg Neg >64 >64
Neg Neg 41 <2 2 4 Neg Neg 64 >64 Neg Neg 42 <2 2 4 Neg Neg
>64 >64 F Neg 43 <2 2 2 Neg Neg >64 >64 F Neg 44
<2 2 2 Neg Neg 64 >64 F Neg 45 <2 2 4 Neg Neg >64
>64 Neg Neg 48 <2 2 2 Neg Neg >64 >64 Neg Neg 47 <2
<2 2 Neg Neg 32 >64 F Neg 48 <2 2 2 Neg Neg >64 >64
F Neg 49 <2 2 2 Neg Neg 64 >64 F Neg CONTROLS 30 <2
NT.sup.b <2 Not >2 4 C,F,R Neg 31 <2 NT <2 Applicable
>2 2 C,F Neg 32 <2 NT <2 2 4 C,F,R Neg 33 <2 NT <2
>2 Died C,D,F,R Tonsil CNS 34 <2 NT <2 >2 4 F Neg
__________________________________________________________________________
.sup.a Clinical signs: NEG = Negative, C = CNS, D = Death, F =
Febrile, R = Respiratory .sup.b Not tested
In this experiment, all of the vaccinated animals remained healthy
following vaccination, developed serum neutralizing antibody to PRV
and did not shed vaccine virus in tonsillar secretions. After
challenge with virulent virus, vaccinates of both age groups
remained free of PRV disease, whereas the 3 non-vaccinated contact
controls and 10 of 11 of the challenge controls developed severe
pseudorabies disease.
Example 7
S-PRV-014
S-PRV-014 is a pseudorabies virus that has a deletion in the gpX
coding region. The gene for E. coli beta-galactosidase was inserted
in place of the gpX gene and is under the control of the endogenous
gpX promoter. The following procedures were used to create
S-PRV-014 by homologous recombination. The flanking PRV homology
regions were from the cloned BamHI #10 fragment which contains the
gpX promoter, and from the cloned BamHI #7 fragment extending from
the NdeI site to the BamHI site (FIG. 9A-9E). The NdeI site was
filled in according to the POLYMERASE FILL-IN REACTION, and the
beta-galactosidase gene was inserted between the BamHI #10 and
BamHI #7 fragments. This construct positioned the
beta-galactosidase gene behind the gpX promoter and the gpX poly A
signal sequence with a deletion of almost all of the coding region
of gpX. The plasmid DNA and DNA from wild-type PRV were mixed and
transfected according to the DNA TRANSFECTION FOR GENERATING
RECOMBINANT VIRUS procedure. The recombinant virus was screened and
purified from the transfection stock by the BLUOGAL SCREEN FOR
RECOMBINANT HERPESVIRUS procedure.
The resulting virus from this screen was designated S-PRV-014 and
has been deposited with the ATCC under Accession No. VR 2135. It
contains the beta-galactosidase gene in place of the gpX coding
region as determined by PREPARATION OF HERPESVIRUS DNA followed by
SOUTHERN BLOTTING DNA. The expression of the beta-galactosidase
gene was confirmed by the BLUOGAL SCREEN FOR RECOMBINANT
HERPESVIRUS test, and by the o-nitro-phenylgalactopyranoside
substrate assay (27). The structure of this virus is shown in FIG.
9D.
Example 8
S-PRV-016
S-PRV-016 is a pseudorabies virus that has a deletion in both
repeat sequences, and a deletion in the gpX coding region. The gene
for E. coli beta-galactosidase was inserted in place of the gpX
gene and is under the control of the endogenous gpX gene
promoter.
The following procedures were used to create S-PRV-016 by
homologous recombination. The flanking PRV homology regions were
from the cloned BamHI #10 fragment which contains the gpX promoter,
and from the cloned BamHI #7 fragment extending from the NdeI site
to the BamHI site (FIG. 9A-9E). The NdeI site was filled in
according to the POLYMERASE FILL-IN REACTION, and the
beta-galactosidase gene was inserted between the BamHI #10 and
BamHI #7 fragments. The construct positioned the beta-galactosidase
gene behind the gpX promoter and the gpX poly A signal sequence
with a deletion of almost all of the coding region of gpX. The
plasmid DNA and DNA from S-PRV-001 were mixed and transfected
according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS
procedure. The recombinant virus was screened and purified from the
transfection stock by the BLUOGAL SCREEN FOR RECOMBINANT
HERPESVIRUS procedure.
The resulting virus from this screen was designated S-PRV-016 and
has been deposited with the ATCC Accession No. VR 2136. It contains
the beta-galactosidase gene in place of the gpX coding region as
determined by PREPARATION OF HERPESVIRUS DNA followed by SOUTHERN
BLOTTING OF DNA. The expression of the beta-galactosidase gene was
confirmed by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS test,
and by the o-nitro-phenylgalactopyranoside substrate assay (27).
The structure of this virus is shown in FIG. 9E.
Example 9
S-PRV-020
S-PRV-020 is a pseudorabies virus that contains a deletion in the
TK gene, a deletion in the repeat regions, and a deletion of the
gpX gene, with an insertion of the swine parvovirus B capsid
protein gene into the gpX region.
For cloning the swine parvovirus B gene, the NADL-8 strain
double-stranded replicative-form DNA was purified from swine
parvovirus infected cells and was supplied by Dr. T. Molitor,
University of Minnesota. The parvovirus NADL-8 DNA was cloned into
the E coli plasmid pSP64 by methods detailed in (15). The DNA was
partially sequenced to allow the determination of the start and the
end of the major caspid protein gene, the B gene. Identification
was confirmed by comparison of related sequences in the rat HI
parvovirus capsid gene (29,30). The sequence of the swine
parvovirus B gene is shown in Fig. 11A and 11B.
The PRV glycoprotein X (gpX) gene promoter was used to express the
B gene and the gpX poly A signal sequence was used to terminate
transcription. The parvovirus B gene from the AccI site at
nucleotide #391 to the RsaI site at nucleotide #2051 was cloned
between the BamHI and NdeI site of gpX (see FIG. 7A and B). Plasmid
pSY864 contained this fragment of the parvovirus B gene flanked by
the gpX signal sequence as shown in FIGS. 13A-13C It was used as
the homologous DNA to promote homologous recombination between
S-PRV-013 DNA and the plasmid DNA to facilitate the incorporation
of the B gene into the PRV genome. The plasmid DNA and S-PRV-013
DNA were mixed and transfected together according to the DNA
TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure. The
recombinant virus was screened and purified for the transfection
stock by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure
with the following modification. Because the parental S-PRV-013
contained the beta-galactosidase gene, it generated blue plaques in
the screening procedure. Since the parvovirus B gene would replace
the beta-galactosidase gene in the virus, the plaques with this
insert would appear colorless. Therefore, colorless plaques were
picked and analyzed during this screening. A virus that contained
the B gene was isolated from this screening and was designated
S-PRV-020. S-PRV-020 has been deposited with the ATCC under
Accession No. VR 2137.
DNA from S-PRV-020 was isolated by the PREPARATION OF HERPESVIRUS
DNA procedure and used to confirm the insertion of the parvovirus B
gene according to the SOUTHERN BLOTTING OF DNA procedure using the
B gene as a probe. The test showed that the parvovirus B gene has
been incorporated into the PRV genome as expected. The structure of
S-PRV-020 is shown in FIG. 13C.
Example 10
S-PRV-025
The cloning of the B gene and construction of these signal
sequences onto the B gene are described in Example 9 and are shown
in FIG. 12.
The DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT
HERPESVIRUS was used to insert the parvovirus B gene into PRV. The
plasmid pSY957 containing the B gene was mixed with S-PRV-002 DNA
and they were cut with restriction enzyme XbaI. The DNA mixture was
ligated as described in the method and the DNA was transfected into
Vero cells. A virus that contained the B gene was isolated from the
transfection stock of virus and was designated S-PRV-025. S-PRV-025
has been deposited with the ATCC under Accession No. VR 2138.
DNA from S-PRV-025 was isolated by the PREPARATION OF HERPESVIRUS
DNA procedure and used to confirm the insertion of the parvovirus B
gene according to the SOUTHERN BLOTTING OF DNA procedure using the
B gene a probe. The test showed that the parvovirus B gene has been
incorporated into the PRV genome as expected. The structure of
S-PRV-025 is shown in FIG. 14A-14C.
Example 11
S-PRV-029
S-PRV-029 is a pseudorabies virus that has a deletion in the
junction region between the unique long region and the internal
repeat of PRV, and a deletion in the gpX gene in the unique short
region. The E. coli beta-galactosidase gene under the control of
the gpX promoter and polyadenylation signals has been inserted into
both deletions in S-PRV-029.
To construct this virus, the SalI #1 fragment of PRV was first
cloned. PRV DNA was prepared and then cut with SalI restriction
enzyme. The cut DNA was electrophoresed on an agarose gel and the
largest SalI band (15 kb) was purified from the gel (see PHENOL
EXTRACTION OF DNA FROM AGAROSE). The purified DNA was ligated into
the plasmid pSP64 (see LIGATION) and the DNA mixture was used to
transform E. coli HB101 according to Maniatis et al. (1). The SalI
#1 clone was mapped for restriction sites.
The homologous recombination procedure was used to create
S-PRV-029. The exact position of the junction region was determined
by sequencing the DNA from the SalI #1 fragment. It was found that
the junction region was positioned between two StuI sites (see FIG.
15B). Two fragments of DNA from the SalI clone were used to create
the homology vector for recombination. One was a fragment from
BamHI #8', from StuI to BamHI and the other was from BamHI to StuI
(FIG. 15B).
The E. coli beta-galactosidase gene was previously engineered to
contain the gpX promoter and polyadenylation signals as described
for S-PRV-013. To put this B-galactosidase gene into the junction
region clone, a HindIII linker was first inserted into the StuI
site between the BamHI #8 and BamHI #8', and into this HindIII site
was cloned a HindIII fragment containing the beta-galactosidase
gene with the gpX signals.
The resulting plasmid plus wild-type PRV DNA were transfected into
Vero cells by the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS
procedure. A virus was isolated from the transfection stock that
contained the beta-galactosidase gene inserted into both the
junction deletion (FIG. 15B) and the gpX deletion (FIG. 15A) due to
the presence of homology to both of these regions in the plasmid.
This virus was purified by the BLUOGAL SCREEN FOR RECOMBINANT
HERPESVIRUS procedure and was designated S-PRV-029. S-PRV-029 has
been deposited with the ATCC under Accession No. VR 2139.
S-PRV-029 was shown to be expressing beta-galactosidase by the
BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure and the
o-nitrophenylgalactopyranoside assay (27). The structure of this
virus is shown in FIG. 15C.
Example 12
S-IBR-002
S-IBR-002 is an IBR virus that has a deletion of approximately 800
bp in the repeat region of the genome. This deletion removes the
only two EcoRV restriction sites on the virus genome and an
adjacent BglII site (FIG. 16).
To construct this virus, the DIRECT LIGATION PROCEDURE FOR
GENERATING RECOMBINANT HERPESVIRUSES was performed. Purified IBR
DNA (Cooper strain) digested with EcoRV restriction enzyme was
mixed with DraI-restriction enzyme-digested plasmid DNA containing
the beta-galactosidase gene under the control of the HSV-1 TK
promoter. After ligation the mixture was used to transfect animal
cells and the transfection stock was screened for recombinant IBR
virus by the HYBRIDIZATION SCREEN FOR RECOMBINANT HERPESVIRUSES
procedure. The final result of the purification was the recombinant
IBR designated S-IBR-002. It was shown by Southern hybridization
that this virus does not carry any foreign genes. Restriction
enzyme analysis also showed that the insertion sites (EcoRV) at
both repeats were deleted. FIG. 16 shows the restriction map of the
EcoRI B fragment which contains the EcoRV restriction sites and the
map of S-IBR-002 which lacks the EcoRV sites. S-IBR-002 has been
deposited with the ATCC under Accession No. VR 2140.
Example 13
S-IBR-004
S-IBR-004 is an IBR recombinant virus carrying an inserted foreign
gene, Tn5 NEO (aminoglycoside 3'-phosphotransferase) gene, under
the control of the pseudorabies virus (PRV) glycoprotein X
promoter.
To construct this virus, the HindIII K DNA fragment from wild type
IBR virus was cloned into the plasmid pSP64 at the HindIII site.
This plasmid was designated pSY524. A map of the HindIII K fragment
is shown in FIG. 17. The DNA from the XhoI site to the HindIII site
and containing the NdeI site from pSY524 was cloned into plasmid
pSP65 and called pSY846. The NdeI to EcoRI fragment was removed
from pSY846 by digestion with NdeI and EcoRI restriction enzymes,
followed by POLYMERASE FILL-IN REACTION and LIGATION. The resulting
plasmid was called pSY862. The plasmid pNEO (P.L. Biochemicals,
Inc.) contains the aminoglycoside 3'-phosphotransferase (NEO) gene
and confers resistance to ampicillin and neomycin on E. coli hosts.
The coding region of this gene (BglII-BamHI fragment) was isolated
and cloned between the PRV gpX promoter and the HSV-Tk poly A
sequence in a plasmid called pSY845.
The NEO gene construct in pSY845 was excised with HindIII, made
blunt ended by the POLYMERASE FILL-IN REACTION, and cloned into the
SacI site of plasmid pSY862. The final product was called
pSY868.
Wild type IBR DNA was mixed with pSY868 DNA and the mixture was
transfected into rabbit skin cells to generate recombinant IBR. The
recombinant IBR virus carrying a functional NEO gene was then
isolated and purified according to the SELECTION OF G418 RESISTANT
VIRUS method.
S-IBR-004 recombinant IBR was shown to express the NEO gene by the
fact that cells infected with this virus were resistant to the
toxicity of G418. A detailed map of the plasmid construction is
shown in FIG. 17. The structure of S-IBR-004 is also shown in FIG.
17. S-IBR-004 has been deposited on May 23, 1986 with the ATCC
under Accession No. VR 2134.
Example 14
S-IBR-008
S-IBR-008 is an IBR virus that has a deletion in the short unique
region, and an insertion of the bovine rotavirus glycoprotein 38
(gp38) gene in the XbaI site in the long unique region.
First the bovine rotavirus gp38 gene was engineered to contain
herpesvirus regulatory signals as shown in FIG. 18. This was
accomplished by cloning the gp38 gene BamHI fragment contained in
pSY1053 between the BamHI and BglII sites in pSY1052. The resulting
plasmid, pSY1023, contained the PRV gpX promoter in front of the
gp38 gene, and the HSV-1 TK polyadenylation signal behind the gp38
gene. The entire construct was flanked by XbaI sites to allow for
the insertion of the XbaI fragment into IBR by direct ligation.
S-IBR-004 was the starting virus for the generation of S-IBR-008.
SIBR-004 DNA and pSY1023 DNA were mixed together, cut with XbaI,
and transfected into rabbit skin cells according to the DIRECT
LIGATION FOR GENERATING RECOMBINANT HERPESVIRUS procedure. The
transfection stock was screened for recombinant virus by the
ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS procedure using
antibodies prepared against the rotavirus gp38 protein.
One of the viruses purified by this screen was S-IBR-008, which has
the following characteristics. It contains the rotavirus gp38 gene
plus the plasmid DNA inserted into the XbaI site in the long unique
region of the virus genome, but no longer contains the NEO gene of
parent S-IBR-004 in the unique short region. In fact, a small
deletion was created in the unique short region at the location of
the NEO gene, as evidenced by the absence of an XbaI site at this
location in S-IBR-008.
S-IBR-008 was shown to be expressing the rotavirus gp38 gene by
analysis of RNA transcription in infected cells, and by the
ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS procedure using
antibodies specific for the gp38 gene. S-IBR-008 has been deposited
with the ATCC under Accession No. VR 2141, and its structure is
shown in FIG. 18.
Example 15
Herpes Virus of Turkeys
Herpes virus of turkeys (HVT) is another herpesvirus that is
similar in organization and structure to the other animal
herpesvirus examples described above. The restriction enzyme map of
HVT has been published (36). This information was used as a
starting point to engineer the insertion of foreign genes into HVT.
The BamHI restriction map of HVT is shown in FIG. 19A. From this
data, several different regions of HVT DNA into which insertions of
foreign genes could be made were targeted. The foreign gene chosen
for insertion was the E. coli beta-galactosidase gene (beta-gal),
which we have used in PRV. The promoter was the PRV gpX promoter.
The beta-gal gene was inserted into the unique long region of HVT,
specifically into the XhoI site in the BamHI #16 (3300bp) fragment,
and has been shown to be expressed in an HVT recombinant by the
formation of blue plaques using the substrate Bluogal. Similarly,
the beta-gal gene has been inserted into the SalI site in the
repeat region contained within the BamHI #19 (900 bp) fragment.
These experiments show that HVT is amenable to the procedures
described within this application for the insertion and expression
of foreign genes in herpesviruses. In particular, two sites for
insertion of foreign DNA have been identified (FIGS. 19B and
19C).
Example 16
Methods for Constructing An Attenuated Herpesvirus Containing A
Foreign DNA Insert
Applicants contemplate that the procedures disclosed herein which
have been utilized to attenuate and insert foreign DNA sequences
into PRV, IBR and HVT may be suitable for constructing other
herpesviruses which are attenuated or contain inserted foreign DNA
sequences which are translated into amino acid sequences in a host
or both. Equine herpesvirus-l (EHV) , canine herpesvirus-l (CHV),
feline herpesvirus-l (FHV) or any animal herpesvirus whose genomic
structure is related to these viruses are contemplated to be
amenable to these methods. More specifically, the following
procedures may be followed to construct such viruses.
GROW ANIMAL HERPESVIRUS IN CELL CULTURE. Established cell lines or
primary cells may be used. The methodology for the growth of these
viruses exists in the literature and does not require new art. EHV
grows in Vero cells, CHV grows in Madin Darby canine kidney cells
and FHV grows in Crandell feline kidney cells.
PURIFY HERPESVIRUS DNA. The procedure disclosed herein for
purifying herpesvirus DNA was successful for all herpesviruses
tested, including PRV, IBR, HVT and cytomegalovirus, and is a
general method applicable to all herpesviruses.
CLONE RESTRICTION FRAGMENTS. The cloning of herpesvirus restriction
fragments is a state of the art recombinant DNA procedure and is
described in Maniatis et al. (1).
MAP RESTRICTION FRAGMENTS TO GENOME. It is useful to have a
restriction enzyme map of the virus genome to identify and select
regions for deletion and insertion. Such maps are available for PRV
and IBR, and partially for HVT. A map exists for EHV, but not for
CHV or FHV. The creation of this map does not require any new
technology and is detailed in Maniatis et al. (1).
IDENTIFY RESTRICTION FRAGMENTS THAT CORRESPOND TO THE REPEAT
REGION. The identification of repeat regions requires the SOUTHERN
BLOTTING PROCEDURE as detailed in the methods section. Clones of
the repeat region hybridize to multiple bands in a restriction
enzyme digest due to the fact that they are repeated in the virus
genome. This feature, coupled with their location in the genome,
are diagnostic of repeat regions.
MAKE DELETION IN REPEAT REGION CLONE. Genetic information in the
repeat region is duplicated in the other copy of the repeat in the
genome. Therefore one copy of the repeat region is nonessential for
replication of the virus. Hence the repeat region is suitable for
deletions and insertions of foreign DNA. After the repeat region is
cloned and mapped by restriction enzymes, enzymes may be chosen to
engineer the repeat deletion and to insert foreign DNA. It is
obvious to one skilled in the art that enzyme sites will exist in a
given stretch of DNA and that they can be found by analysis. The
methodology involves RESTRICTION DIGESTION OF DNA, AGAROSE GEL
ELECTROPHORESIS OF DNA, LIGATION and cloning in bacterial cells as
detailed in the methods section and in Maniatis et al. (1).
MAKE INSERTION OF MARKER GENE INTO DELETION IN REPEAT REGION CLONE.
The methodology of this insertion is that described in Maniatis et
al. (1) for the cloning of genes into bacteria. What is not obvious
prior to the present disclosure is which marker genes to use that
will be active in a herpesvirus, nor which signal sequences to use
for the expression of foreign genes in these herpesviruses. The E.
Coli beta-galactosidase gene and neomycin resistance gene under the
control of the HSV-1 ICP4 promoter, the PRV gpX promoter or the
HSV-1 TK promoter have been used. The gpX promoter, in particular,
works in PRV, IBR, and HVT. The other promoters have also worked in
more limited testing.
TRANSFECTION WITH MARKER GENE CLONE+HERPESVIRUS DNA. The intent of
this procedure is to put into the same cell the intact herpesvirus
DNA and the repeat region clone with the deletion and containing
the marker gene. Once these two DNAs are present in the same cell,
normal mechanisms of homologous recombination ensure that a
recombination will occur between the homologous regions in the
clone and the same region in the herpesvirus DNA, thus substituting
the marker gene for the deleted regions in the virus, with
frequency of about 1%. The technique involves the TRANSFECTION
PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSES as detailed in
the methods section.
PURIFY HERPESVIRUS DNA. Herpesvirus DNA may be purified according
to the methods described above.
SELECT RECOMBINANT PLAQUE. All the herpesviruses contemplated by
this invention form plaques (foci of infection in cell culture)
that enable their purification. A plaque results from infection by
a single virus particle. Thus picking a single plaque selects for
the progeny of a single recombinational event. This technical feat
requires a method to identify which plaque to pick. The methods
used herein include SOUTHERN BLOTTING OF DNA to pick the plaque
based upon the presence of the inserted gene, ANTIBODY SCREEN FOR
RECOMBINANT HERPESVIRUS to pick the plaque based upon the presence
of protein made from the gene, BLUOGAL SCREEN FOR RECOMBINANT
HERPESVIRUSES to pick a plaque that expresses the marker gene
beta-galactosidase or G-418 SELECTION TO PURIFY RECOMBINANT
HERPESVIRUSES to pick the plaque by its ability to form in the
presence of the antibiotic G-418. The first two methods are
applicable to any gene; the latter two are specific for the
beta-galactosidase gene and neomycin resistance gene respectively.
The biology of these screening and selection systems is such that
they are applicable to any herpesvirus, including EHV, CHV, FHV,
and any animal herpesvirus related to them.
PURIFY RECOMBINANT VIRUS. This procedure involves multiple plaque
purifications in succession to completely purify the recombinant
virus away from the parental virus. The screening is applied at
each step to choose the plaque with which to continue. The
procedures are known to those skilled in the art of virology.
Multivalent vaccines for animals may be constructed by inserting a
foreign antigen gene into a herpesvirus. The procedures and
methodology are very analogous to those used for the initial
insertion of the marker gene into the virus and may be performed as
follows.
SUBSTITUTE FOREIGN ANTIGEN GENE FOR MARKER GENE IN REPEAT CLONE .
This is a cloning experiment that involves putting the antigen gene
behind the same herpesvirus promoter used with the marker gene and
inserting this construction into the same identical deletion in the
repeat clone. The methods for this cloning are described in
Maniatis et al. (1).
TRANSFECTION WITH ANTIGEN CLONE+RECOMBINANT HERPES DNA CONTAINING
MARKER. The marker gene that is already present in the herpesvirus
genome may be used to aid in the selection of the new recombinant.
For example, it has proven useful to select white plaques instead
of blue ones to test for the absence of beta-galactosidase in this
step. One reason for the present of a white plaque is the
replacement of the beta-galactosidase gene with the foreign antigen
gene by homologous recombination (the desired outcome). Continued
screening for this new recombinant by the SOUTHERN BLOT PROCEDURE
or by the ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS becomes more
focused, less time-consuming and specifically identifies the
recombinant of interest.
ISOLATE RECOMBINANT HERPES DNA. The transfection procedure requires
intact infectious herpesvirus DNA. Recombinant herpesviruses which
include an inserted marker gene may be used. The isolation of
herpesvirus DNA procedure is equally applicable to these
recombinant viruses.
SCREEN TRANSFECTION STOCK FOR FOREIGN GENE INSERTION. Screening
methods have been described above. They are a combination of
indirect methods (screening for the absence of the marker) as well
as direct methods (SOUTHERN BLOT for the antigen gene, and ANTIBODY
SCREEN for the expressed protein). The methods may be applied
sequentially during the purification of the virus.
PURIFY RECOMBINANT VIRUSES CONTAINING FOREIGN ANTIGEN GENE. The
recombinant virus containing the foreign antigen gene may be
purified according to the procedures described above.
This sequence of steps, along with the methods and examples
described herein, enable anyone skilled in the art to successfully
practice this invention with any animal herpesvirus.
Example 17
The present invention involves the use of genetically engineered
herpesviruses to protect animals against disease. It was not
apparent at the outset of research which deletions in herpesviruses
would serve to attenuate the viruses to the proper degree so as to
render them useful as vaccines. Even testing vaccine candidates in
animal models, e.g. mouse, does not serve as a valid indicator of
the safety and efficacy of the vaccine in the target animal
species, e.g. swine. To illustrate this point more clearly, Table
VII shows summary data of the safety and efficacy of various
pseudorabies viruses which were constructed and tested in swine
according to the VACCINATION STUDIES IN SWINE procedure.
TABLE VII ______________________________________ SUMMARY OF STUDIES
CONDUCTED IN PIGS WITH VARIOUS PSEUDORABIES VIRUS CONSTRUCTS
Percent Construct Age Post-Vaccination Protection (Deletions/
Number of Antibody Clinical Against Insertions).sup.1 of Pigs Pigs
Range Signs Challenge ______________________________________
S-PRV-001 9 4-6 1:32- Yes Not Done (A) weeks >1:64 (22%)
S-PRV-002 12 4-6 1:4- None 100 (A,B) weeks 1:64 S-PRV-003 8 4-6
<1:2- None 50 (B) weeks 1:16 S-PRV-004 6 4-6 1:4- None 64 (B,C)
weeks 1:32 S-PRV-010 30 4-6 <1:2- Yes 100 (A,B,E) weeks 1:16
(13%) 30 3-4 1:4- Yes 100 days 1:64 (13%) S-PRV-013 23 4-6 <1:2-
None 100 (A,B,D,E) weeks 1:8 25 3-4 1:4- None 100 days 1:64
S-PRV-014 5 4-6 1:4- Yes 100 (D,E) weeks 1:8 (40%) S-PRV-016 5 4-6
1:4- None 100 (A,D,E) weeks 1:8
______________________________________ .sup.1 ARepeats; BTK;
CJunction; DgpX; Ebeta-galactosidase insert
The eight constructs that have been tested have the following
deletions and insertions in the genome of the virulent Shope strain
of PRV: S-PRV-001 has a deletion in both repeat regions; S-PRV-002
has a deletion in both repeat regions and in the thymidine kinase
gene; S-PRV-003 has a deletion in the thymidine kinase gene;
S-PRV-004, S-PRV-010, S-PRV-013, S-PRV-014 and S-PRV-016 are
described in Example #'s 1, 3, 6, 7 and 8 respectively.
A superior vaccine product must not produce clinical signs in 3-4
day old piglets (the more sensitive age), and give 100% protection
in pigs of all ages. From Table VII, it is apparent that each
vaccine candidate provided some degree of attenuation and
protection in swine, but each vaccine provided a unique response.
The utility of the subject combinations of genomic deletions and
foreign DNA insertions was unexpected and the resulting attenuated
pseudorabies viruses are both novel and useful as pseudorabies
vaccines.
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